Comparative Genomic Hybridization (CGH)

ABSTRACT

Disclosed are new methods comprising the use of in situ hybridization to detect abnormal nucleic acid sequence copy numbers in one or more genomes wherein repetitive sequences that bind to multiple loci in a reference chromosome spread are either substantially removed and/or their hybridization signals suppressed. The invention termed Comparative Genomic Hybridization (CGH) provides for methods of determining the relative number of copies of nucleic acid sequences in one or more subject genomes or portions thereof (for example, a tumor cell) as a function of the location of those sequences in a reference genome (for example, a normal human genome). The intensity(ies) of the signals from each labeled subject nucleic acid and/or the differences in the ratios between different signals from the labeled subject nucleic acid sequences are compared to determine the relative copy numbers of the nucleic acid sequences in the one or more subject genomes as a function of position along the reference chromosome spread. Amplifications, duplications and/or deletions in the subject genome(s) can be detected. Also provided is a method of determining the absolute copy numbers of substantially all RNA or DNA sequences in subject cell(s) or cell population(s).

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 08/223,905,filed on Apr. 6, 1994 now abandoned, which is a continuation ofapplication Ser. No. 08/132,172, filed on Oct. 6, 1993 now abandoned,which is a continuation-in-part of application Ser. No. 07/969,948,filed on Oct. 20, 1992 (now abandoned), which is a continuation-in-partof application Ser. No. 07/846,659 filed on Mar. 4, 1992, (nowabandoned).

FIELD OF THE INVENTION

This invention relates generally to the field of cytogenetics, and moreparticularly to the field of molecular cytogenetics. It concerns methodsof determining the relative copy numbers of different nucleic acidsequences in a subject cell or cell population and/or comparing thenucleic acid sequence copy numbers of substantially identical sequencesin several cells or cell populations as a function of the location ofthose sequences in a reference genome. For instance, the methods of thisinvention provide the means to determine the relative number of copiesof nucleic acid sequences in one or more subject genomes (for example,the DNA of one tumor cell or a number of cells from a subregion of asolid tumor) or portions thereof as a function of the location of thosesequences in a reference genome (for example, a normal human metaphasespread). Further, the invention provides methods of determining theabsolute copy number of nucleic acid sequences in a subject cell or cellpopulation.

Although the examples herein concern human cells and the language isprimarily directed to human concerns, the concept of this invention isapplicable to genomes from any plant or animal. The genomes comparedneed only be related closely enough to have sufficient substantiallyidentical sequences for a meaningful analysis. For example, a humangenome and that of another primate could be compared according to themethods of this invention.

BACKGROUND OF THE INVENTION

Chromosome abnormalities are associated with genetic disorders,degenerative diseases, and exposure to agents known to causedegenerative diseases, particularly cancer, German, "Studying HumanChromosomes Today," American Scientist. 58: 182-201 (1970); Yunis; "TheChromosomal Basis of Human Neoplasia," Science. 221: 227-236 (1983); andGerman, "Clinical Implication of Chromosome Brealkge," in Genetic Damagein Man Caused by Environmental Agents, Berg, Ed., pgs. 65-86 (AcademicPress, New York, 1979). Chromosomal abnormalities can be of severaltypes, including: extra or missing individual chromosomes, extra ormissing portions of a chromosome (segmental duplications or deletions),breaks, rings and chromosomal rearrangements, among others. Chromosomalor genetic rearrangements include translocations (transfer of a piecefrom one chromosome onto another chromosome), dicentrics (chromosomeswith two centromeres), inversions (reversal in polarity of a chromosomalsegment), insertions, amplifications, and deletions.

Detectable chromosomal abnormalities occur with a frequency of one inevery 250 human births. Abnormalities that involve deletions oradditions of chromosomal material alter the gene balance of an organismand generally lead to fetal death or to serious mental and physicaldefects. Down syndrome can be caused by having three copies ofchromosome 21 instead of the normal 2. This syndrome is an example of acondition caused by abnormal chromosome number, or aneuploidy. Downsyndrome can also be caused by a segmental duplication of a subregion onchromosome 21 (such as, 21q22), which can be present on chromosome 21 oron another chromosome. Edward syndrome (18+), Patau syndrome (13+),Turner syndrome (XO) and Kleinfelter syndrome (XXY) are among the mostcommon numerical aberrations. [Epstein, Ihe Consequences of ChromosomeImbalance: Principles, Mechanisms and Models (Cambridge Univ. Press1986); Jacobs, Am. J. Epidemiol. 105: 180 (1977); and Lubs et al.,Science. 169; 495 (1970).]

Retinoblastoma (del 13q14), Prader-Willis syndrome (del 15q11-q13),Wilm's tumor (del 11p13) and Cri-du-chat syndrome (del 5p) are examplesof important disease linked structural aberrations. [Nora and Fraser,Medical Genetics: Prnciples and Practice, (Lea and Febiger (1989).]

One of the critical endeavors in human medical research is the discoveryof genetic abnormalities that are central to adverse healthconsequences. In many cases, clues to the location of specific genesand/or critical diagnostic markers come from identification of portionsof the genome that are present at abnormal copy numbers. For example, inprenatal diagnosis, as indicated above, extra or missing copies of wholechromosomes are the most frequently occurring genetic lesion. In cancer,deletion or multiplication of copies of whole chromosomes or chromosomalsegments, and higher level amplifications of specific regions of thegenome, are common occurrences.

Much of such cytogenetic information has come over the last severaldecades from studies of chromosomes with light microscopy. For the pastthirty years cytogeneticists have studied chromosomes in malignant cellsto determine sites of recurrent abnormality to glean hints to thelocation of critical genes. Even though cytogenetic resolution islimited to several megabases by the complex packing of DNA into thechromosomes, this effort has yielded crucial information. Among thestrengths of such traditional cytogenetics is the ability to give anoverview of an entire genome at one time, permitting recognition ofstructural abnormalities such as inversions and translocations, as wellas deletions, multiplications, and amplifications of whole chromosomesor portions thereof. With the coming of cloning and detailed molecularanalysis, recurrent translocation sites have been recognized as involvedin the formation of chimeric genes such as the BCR-ABL fusion in chronicmyelogeneous leukemia (CML); deletions have been recognized asfrequently indicating the location of tumor suppressor genes; andamplifications have been recognized as indicating overexpressed genes.

Conventional procedures for genetic screening and biological dosimetryinvolve the analysis of karyotypes. A karyotype is the particularchromosome complement of an individual or of a related group ofindividuals, as defined both by the number and morphology of thechromosomes usually in mitotic metaphase. It include such things astotal chromosome number, copy number of individual chromosome types(e.g., the number of copies of chromosome X), and chromosomalmorphology, e.g., as measured by length, centromeric index,connectedness, or the like. Karyotypes are conventionally determined bychemically staining an organism's metaphase, prophase or otherwisecondensed (for example, by premature chromosome condensation)chromosomes. Condensed chromosomes are used because, until recently, ithas not been possible to visualize interphase chromosomes due to theirdispersed condition and the lack of visible boundaries between them inthe cell nucleus.

A number of cytological techniques based upon chemical stains have beendeveloped which produce longitudinal patterns on condensed chromosomes,generally referred to as bands. The banding pattern of each chromosomewithin an organism usually permits unambiguous identification of eachchromosome type (Latt, "Optical studies of Metaphase ChromosomeOrganization," Annual Review of Biophysics and Bioengineering. 5: 1-37(1976)].

Unfortunately, such conventional banding analysis requires cellculturing and preparation of high quality metaphase spreads, which istime consuming and labor intensive, and frequently difficult orimpossible. For example, cells from many tumor types are difficult toculture, and it is not clear that the cultured cells are representativeof the original tumor cell population. Fetal cells capable of beingcultured, need to be cultured for several weeks to obtain enoughmetaphase cells for analysis. Over the past decade, methods of in situhybridization have been developed that permit analysis of intact cellnuclii-interphase cytogenetics. Probes for chromosome centromeres, wholechromosomes, and chromosomal segments down to the size of genes, havebeen developed. With the use of such probes, the presence or absence ofspecific abnormalities can be very efficienty determined; however, it istedious to test for numerous possible abnormalities or to survey todiscover new regions of the genome that are altered in a disease.

The present invention, Comparative Genomic Hybridization (CGH) [formerlycalled Copy Ratio Reverse Cytogenetics (CRRC) among other names]provides powerful methods to overcome many of the limitations ofexisting cytogenetic techniques. When CGH is applied, for example, inthe fields of tumor cytogenetics and prenatal diagnosis, it providesmethods to determine whether there are abnormal copy numbers of nucleicacid sequences anywhere in the genome of a subject tumor cell or fetalcell or the genomes from representative cells from a tumor cellpopulation or from a number of fetal cells, without having to preparecondensed chromosome spreads from those cells. Thus, cytogeneticabnormalities involving abnormal copy numbers of nucleic acid sequences,specifically amplifications and/or deletions, can be found by themethods of this invention in the format of an immediate overview of anentire genome or portions thereof. More specifically, CGH providesmethods to compare and map the frequency of nucleic acid sequences fromone or more subject genomes or portions thereof in relation to areference genome. It permits the determination of the relative number ofcopies of nucleic acid sequences in one or more subject genomes (forexample, those of tumor cells) as a function of the location of thosesequences in a reference genome (for example, that of a normal humancell).

Gene amplification is one of several mechanisms whereby cells can changephenotypic expression when increased amounts of specific proteins arerequired, for example, during development [Spradling and Mahowald, PNAS(USA), 77: 1096-1100 (1980); Glover et al., PNAS (USA 79; 2947-2951(1982), or during an environmental challenge when increased amounts ofspecific proteins can impart resistance to cytotoxic agents [Melera etal., J. Biol. Chem. 255: 7024-7028 (1980); Beach and Palmiter, PNAS(USA, 78: 2110-2114 (1981)].

A major limitation of Southern analysis and related conventionaltechniques for analysis of gene amplification is that only specificsites are studied leaving the vast majority of the genome unexamined.Conventional cytogenetic studies, on the other hand, provide a broadsurvey of the genome but provide little information about genes that maybe involved in amplification events. However, the procedures of thisinvention overcome those limitations. This invention can be used to showthe normal chromosomal locations of all regions of a genome that areamplified or deleted wherein the size of the regions that can bedetected is limited only by the resolution of the microscopy used andthe organization of DNA in condensed chromosomes. Thus, this inventionprovides among other uses the ability to study gene amplifications anddeletions and their roles in tumor development, progression and responseto therapy more thoroughly than was possible previously. The methods ofCGH are sufficiently rapid and simple that large numbers of subjectnucleic acids, for example from many tumors, can be analysed in studiesfor gene amplification and deletion.

The karyotypic heterogeneity in solid tumors can be extreme.Identification of commonly occurring chromosomal changes by analysis ofmetaphase spreads is often difficult or impossible using conventionalbanding analysis because of the complexity of the rearrangements andbecause of the poor quality of the metaphase preparations. CGH overcomesthat limitation in that the tumor nucleic acid can be studied withoutthe requirement of preparing metaphase spreads. Since CGH can probablybe performed on single cells by amplifying the nucleic acid therefrom,CGH can be used to investigate the heterogeneity of tumors by studyingrepresentative cells from different cell populations of the tumor.Alternatively, CGH of nucleic acid from a tumor extracted in a bulkextraction process from many cells of the tumor can reveal consistencieswithin the apparent heterogeneity. For example, the same amplifiedsequences may appear as homogeneously staining regions (HSRS) and/ordouble minute chromosomes (DMs) in one tumor cell but as an extension ofa chromosome arm in another tumor cell. Thus, order from the apparentrandomness may be realized by CGH hybridization.

Montgomery et al., PNAS (USA), 80: 5724-5728 (September 1983), concernsthe hybridization of labeled Cot fractionated DNAs from tumor cell lines(a Cot fraction from which the high copy repeats, low copy repeats andsingle copy sequences were substantially removed) to metaphase spreadsfrom said tumor cell lines. Basically, Montgomery et al. mapped thepositions of nucleic acid sequences from tumor cell lines that are veryhighly amplified back to tumor cell line genomes.

Total genomic DNA from one species has been used in in situhybridization to discriminate in hybrid cells between chromosomes ofthat species and of a different species on the basis of the signal fromthe high copy repetitive sequences. [Pinkel et al., PNAS (USA), 83: 2934(1986); Manuelidis, Hum. Genet., 71: 288 (1985); and Durnam et al.,Somatic Cell Molec. Genet., 11: 571 (1985).] Landegent et al., Hum.Genet., 77 366-370 (1987), eliminated highly repetitive sequences, likeAlu and Kpn fragments, from whole cosmid cloned genomic sequences byblocking the highly repetitive sequences with Cot-1 DNA. The resultingprobe was used for in situ hybridization.

European Patent Application Publication No. 430,402 (published Jun. 5,1991) describes methods and compositions for chromosome-specificpainting, that is, methods and compositions for staining chromosomesbased upon nucleic acid sequence employing high complexity nucleic acidprobes. In general in the chromosome-specific painting methods,repetitive sequences not specific to the targeted nucleic acid sequencesare removed from the hybridization mixture and/or their hybridizationcapacity disabled, often by blocking with unlabeled genomic DNA or withDNA enriched for high copy repetitive sequences as is Cot-1[commercially available from Bethesda Research Laboratory, Gaithersburg,Md. (USA)]. Pinkel et al., PNAS (USA), 85; 9138-9142 (1980) alsodescribes aspects of chromosome-specific painting as well asInternational Publication No. WO 90/05789 (published May 31, 1990entitled "in situ Suppression Hybridization and Uses Therefor").

Chromosome-specific repeat sequence probes and chromosome-specificpainting probes can be hybridized in ilih to interphase nuclei as wellas metaphase spreads and provide information about the genetic state ofthe individual targeted genomes. A limitation of such hybridizations isthat cytogenetic information is only provided from the regions to whichthe probes bind. Such hybridizations are very useful for determining ifa particular abnormality is present, for example, the deletion of aspecific gene or a duplication among other abnormalities, but it islaborious to search for currently unknown abnormalities on a region byregion basis.

Other methods of searching for unknown genetic abnormalities similarlyrequire a lot of work. For example, looking for loss of heterozygosityin tumor cells, requires the hybridization of many probes to Southernblots of tumor and normal cell DNA. The instant invention, ComparativeGenomic Hybridization (CGH), provides methods to overcome many of thelimitations of the existing cytogenetic techniques.

Saint-Ruf et al, Genes, Chromosomes & Cancer, 2: 18-26 (1990) state atpage 24 that

Human breast carcinomas are characterized by two sets of molecularanomalies. Firstly, some protooncogenes, such as MYC, INT2, HST, andERBB2, are frequently found either amplified or overexpressed . . . .Secondly, loss of heterozygosity has been reported, especially for ip,11, 13 and 17 . . .

Human breast carcinomas are also characterized cytogenetically byvarious anomalies that may be the chromosomal counterpart of themolecular anomalies: regions of amplification (HSRS) are found in morethan one-third of the tumors . . . and various deletions, affecting,e.g., 1p, 11p, 11q, 13, and 17p, are found recurrently . . . .

[Citations omitted.] Saint-Ruf et al. concluded from the reportedexperiments that although amplification of genetic material is afrequent and probably important event in breast carcinogenesis, that therelevant genes involved in such amplifications remain unknown but do notseem to correspond to the proto-oncogenes commonly considered importantin breast cancer.

Since HSRs in tumors are most often not at the site of the amplifiedgene(s) in normal cells, standard cytogenetics does not yield anyinformation that could assist with identification of the gene(s). CGH onthe other hand permits mapping them in the normal genome, a major steptowards their identification.

Dutrillaux et al., Cancer Genet, Cytogenet., 49: 203-217 (1990) report(at page 203) that "[a]lthough human breast carcinomas are among themost frequent malignant tumors, cytogenctic data remain scarce, probablybecause of their great variability and of the frequent difficulty oftheir analysis." In their study of "30 cases with relatively simplekryotypes to determine which anomalies occur the most frequently and, inparticular, early during tumor progression" (p. 203), they concludedthat "trisomy iq and monosomy 16q are early chromosomal changes inbreast cancer, whereas other deletions and gain of gq are clearlysecondary events." [Abstract, p. 203.] Dutrillaux et al. further state(at page 216) that deletions within tumor suppressor genes "characterizetumor progression of breast cancer."

It is believed that many solid tumors, such as breast cancer, progressfrom initiation to metastasis through the accumulation of severalgenetic aberrations. [Smith et al., Breast Cancer Res. Treat., 18 Suppl.1: S 514 (1991); van de Vijver and Nusse, Biochim. Biophys. Acta,1072:33-50 (1991); Sato et al., Cancer Res., 7184-7189 (1990).] Suchgenetic aberrations, as they accumulate, may confer proliferativeadvantages, genetic instability and the attendant ability to evolve drugresistance rapidly, and enhanced angiogenesis, proteolysis andmetastasis. The genetic aberrations may affect either recessive "tumorsuppressor genes" or dominantly acting oncogenes. Deletions andrecombination leading to loss of heterozygosity (LOH) are believed toplay a major role in tumor progression by uncovering mutated tumorsuppressor alleles.

Dominantly acting genes associated with human solid tumors typicallyexert their effect by overexpression or altered expression. Geneamplification is a common mechanism leading to upregulation of geneexpression. [Stark et al., Cell. 75: 901-908 (1989).] Evidence fromcytogenetic studies indicates that significant amplification occurs inover 50% of human breast cancers. [Saint-Ruf et al., supra.] A varietyof oncogenes have been found to be ampliied in human malignancies.Examples of the amplification of cellular oncogenes in human tumors isshown in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Amplified              Degree of  DM or HSR                                                                      Gene Tumor Amplification Present           ______________________________________                                        c-myc  Promyelocytic leukemia                                                                        20×  +                                              cell line, HL60                                                               Small-cell lung 5-30× ?                                                 carcinoma cell lines                                                         N-myc Primary neuroblastomas 5-1000× +                                   (stages III and IV)                                                           and neuroblastoma                                                             cell lines                                                                    Retinoblastoma cell 10-200× +                                           line and primary                                                              tumors                                                                        Small-cell lung carcinoma 50× +                                         cell lines and tumors                                                        L-myc Small-cell lung carcinoma 10-20× ?                                 cell lines and tumors                                                        c-myb Acute myeloid leukemia 5-10× ?                                     Colon carcinoma cell lines 10× ?                                       c-erbb Epidermoid carcinoma cell 30× ?                                   Primary gliomas  ?                                                           c-K-ras-2 Primarycarcinomas of lung, 4-20× ?                             colon, bladder, and rectum                                                   N-ras Mammary carcinoma cell 5-10× ?                                     line                                                                       ______________________________________                                         SOURCE: modified from Varmus, Ann. Rev. Genetics. 18: 553-612 (1984)          [cited in Watson et al., MoleculaL Biology of the Gene (4th ed.;              Benjamin/Cummings Publishing Co. 1987)                                   

Chromosomal deletions involving tumor suppressor genes may play animportant role in the development and progression of solid tumors. Theretinoblastoma tumor suppressor gene (Rb-1), located in chromosome13q14, is the most extensively characterized tumor suppressor gene(Friend et al., Nature, 323: 643 (1986); Lee et al., Science. 235: 1394(1987); Fung et al., Science, 236: 1657 (1987)]. The Rb-1 gene product,a 105 kDa nuclear phosphoprotein, apparently plays an important role incell cycle regulation [Le et al., supra (1987); Howe et al., PNAS (USA),1 5883 (1990)]. Altered or lost expression of the Rb protein is causedby inactivation of both gene alleles either through a point mutation ora chromosomal deletion. Rb-1 gene alterations have been found to bepresent not only in retinoblastomas [Friend et al., supra (1986); Lee etal., supra (1987); Fung et al., supra (1987)] but also in othermalignancies such as osteosarcomas [Friend et al., supra (1986)], smallcell lung cancer [Hensel et al., Cancer Res., 50; 3067 (1990); Rygaardet al., Cancer Res., 50: 5312 (1990)] and breast cancer [Lee et al.,Science. 241: 218 (1988); T'Ang et al., Science. 242: 263 (1988); Varleyet al., Oncogene. 4: 725 (1989)]. Restriction fragment lengthpolymorphism (RFLP) studies have indicated that such tumor types havefrequently lost heterozygosity at 13q suggesting that one of the Rb-igene alleles has been lost due to a gross chromosomal deletion [Bowcocket al., Am. J. Hum. Genet., 46: 12 (1990)].

The deletion of the short arm of chromosome 3 has been associated withseveral cancers, for example, small cell lung cancer, renal and ovariancancers; it has been postulated that one or more putative tumorsuppressor genes is or are located in the p region of chromosome 3 (ch.3p) [Minna et al., Symposia on Ouantitative Biology, Vol. LI: 843-853(SCH Lab 1986); Cohen et al., N. Eng. J. Med., 301: 592-595 (1979);Bergerham et al., Cancer Res., 49: 1390-1396 (1989); Whang-Peng et al.,Can. Genet. Cytogenet., 11: 91-106 (1984; and Trent et al., Can. Genet.Cytogenet., 14: 153-161 (1985)].

The above-indicated collection of amplified and deleted genes is farfrom complete. As the Saint-Ruf et al. study (supra) of oncogeneamplification in cells showing cytogenetic evidence of amplification,such as double minutes (DMs) or homogeneously staining regions (HSRs),indicated, the amplified genes were not known oncogenes in most cases.As Dutrillaux et al., suora indicated, "cytoglenetic data remainsscarce" for "the most frequent malignant tumors"--breast carcinomas.

Discovery of genetic changes involved in the development of solid tumorshas proven difficult. Karyotyping is impeded by the low yield of highqualJity metaphases and the complex nature of chromosomal changes[Teyssier, J. R., Cancer Genet. Cytogenet., 31: 103 (1989)]. Althoughmolecular genetic studies of isolated tumor DNA have been moresuccessful and permitted detection of common regions of allelic loss,mutation or amplification [Fearon et al., Cell. 61: 759 (1990); Sato etal., Cancer Res., 50: 7184 (1990); Alitalo et al., Adv. Cancer Res., 47:235 (1986); and Schwab and Amler, Genes Chrom. Cancer., 1; 181 (1990)],such molecular methods are highly focused, targeting one specific geneor chromosome region at a time, and leaving the majority of the genomeunexamined.

Thus, a research tool leading to the identification of amplified anddeleted genes and providing more cytogenetic data regarding tumors,especially tumor progression and invasiveness is needed in tumorcytogenetics. CGH provides such a molecular cytogenetic research tool.

CGH facilitates the genetic analysis of tumors in that it provides acopy number karyotype of the entire genome in a single step. Regions oftumor DNA gain and loss are mapped directly onto normal chromosomes.Comparisons of primary tumors with their metastases by CGH should beinformative concerning cancer progression.

The ability to survey the whole genome in a single hybridization is adistinct advantage over allelic loss studies by restriction fragmentlength polymorphism (RLP) that target only one locus at a time. RFLP isalso restricted by the availability and informativeness of polymorphicprobes.

The copy number karyotype determined by CGH may become as important fordiagnostic and/or prognostic assessment of solid tumors as conventionalkaryotyping now is for hematologic malignancies. [Yunis, J. J., Science,221: 227 (1983); Solomon et al., Science, 254: 1153 (1991).]

SUMMARY OF THE INVENTION

Comparative Genomic Hybridization (CGH) employs the kinetics of in situhybridization to compare the copy numbers of different DNA or RNAsequences from a sample, or the copy numbers of different DNA or RNAsequences in one sample to the copy numbers of the substantiallyidentical sequences in another sample. In many useful applications ofCGH, the DNA or RNA is isolated from a subject cell or cell population.The comparisons can be qualitative or quantitative. Procedures aredescribed that permit determination of the absolute copy numbers of DNAsequences throughout the genome of a cell or cell population if theabsolute copy number is known or determined for one or severalsequences. The different sequences are discriminated from each other bythe different locations of their binding sites when hybridized to areference genome, usually metaphase chromosomes but in certain casesinterphase nuclei. The copy number information originates fromcomparisons of the intensities of the hybridization signals among thedifferent locations on the reference genome.

Two representative basic approaches are employed in CGH as illustratedherein for the analysis of subject DNAS. In an example of the firstapproach, genomic DNA from a subject cell or cell population of cells isisolated, labeled and hybridized to reference chromosomes, usually inmetaphase. In an example of the second approach, genomic DNAs from twoor more subject cells or cell populations are isolated, differentiallylabeled, and hybridized to reference chromosomes, usually in metaphase.

The CGH methods of this invention can be qualitative and/orquantitative. A particular utility of CGH is for analysing DNA sequencesfrom subject cell(s) or cell populations, for example from clinicalspecimens including tumor and fetal tissues.

An important utility of CGH is to find regions in normal genomes whichwhen altered in sequence copy number contribute to disease, as forexample, cancer or birth defects. For example, regions at elevated copynumber may contain oncogenes, and regions present at decreased copynumber may contain tumor suppressor genes.

A representative CGH method is for comparing copy numbers of differentDNA sequences in a subject cell or cell population comprising the stepsof:

a) extracting the DNA from the subject cell or from a number of cells ofthe subject cell population;

b) amplifying said extracted subject DNA, if necessary;

c) labeling the subject DNA;

d) hybridizing said labeled subject DNA in situ to reference metaphasechromosomes after substantially removing from the labeled DNA thoserepetitive sequences that could bind to multiple loci in the referencemetaphase chromosomes, and/or after blocking the binding sites for thoserepetitive sequences in the reference metaphase chromosomes byprehybridization with appropriate blocking nucleic acids, and/orblocking those repetitive sequences in the labeled DNA byprehybridization with appropriate blocking nucleic acid sequences,and/or including such blocking nucleic acid sequences for saidrepetitive sequences during said hybridization, wherein the DNAsequences in the labeled subject DNA that bind to single copy sequencesin the reference metaphase chromosomes are substantially retained, andthose single copy DNA sequences as well as their binding sites in thereference metaphase chromosomes remain substantially unblocked bothbefore and during the hybridization;

e) rendering the bound, labeled DNA sequences visualizable, ifnecessary;

f) observing and/or measuring the intensity of the signal from thelabeled subject DNA sequences as a function of position on the referencemetaphase chromosomes; and

g) comparing the copy numbers of different DNA sequences of the subjectDNA by comparing the signal intensities at different positions on thereference metaphase chromosomes, wherein the greater the signalintensity at a given position, the greater the copy number of thesequences in the subject DNA that bind at that position. An analogousmethod can be performed wherein the subject nucleic acid is RNA.

Further, disclosed are methods wherein two or more subject nucleic acidsare analysed by CGH. Exemplary methods are those wherein the subjectnucleic acids are DNA sequences from a subject cell or cell population.Analogous methods may be performed wherein the subject nucleic acids areRNA. Such an exemplary method is that for comparing copy numbers ofdifferent DNA sequences in one subject cell or cell population relativeto copy numbers of substantially identical sequences in another cell orcell population, said method comprising the steps of:

a) extrctg the DNA from both of the subject cells or cell populations;

b) amplifyg said extracted subject DNAs, if necessary;

c) differentially labeling the subject DNAs,

d) hybridizing said differentially labeled subject

DNAs in situ to reference metaphase chromosomes after substantiallyremoving from the labeled DNAs those repetitive sequences that couldbind to multiple loci in the reference metaphase chromosomes, and/orafter blocking the binding sites for those repetitive sequences in thereference metaphase chromosomes by prehybridization with appropriateblocking nucleic acids, and/or blocking those repetitive sequences inthe labeled DNA by prehybridization with appropriate blocking nucleicacid sequences, and/or including such blocking nucleic acid sequencesfor said repetitive sequences during said hybridization;

e) rendering the bound, differentially labeled DNA sequencesvisualizable, if necessary;

f) observing and/or measuring the intensities of the signals from eachsubject DNA, and the relative intensities, as a function of positionalong the reference metaphase chromosomes; and

g) comparing the relative intensities among different locations alongthe reference metaphase chromosomes wherein the greater the intensity ofthe signal at a location due to one subject DNA relative to theintensity of the signal due to the other subject DNA at that location,the greater the copy number of the sequence that binds at that locationin the first subject cell or cell population relative to the copy numberof the substantially identical sequence in the second subject cell orcell population that binds at that location.

Further disclosed are methods of quantitatively comparing copy numbersof different DNA sequences in one subject cell or cell populationrelative to copy numbers of substantially identical sequences in anothersubject cell or cell population. A representative method is thatcomprising steps (a) through (e) of the method immediately detailedabove and the following steps of:

f. measuring the intensities of the signals from each of the boundsubject DNAs and calculating the ratio of the intensities as a functionof position along the reference metaphase chromosomes to form a ratioprofile; and

g. quantitatively comparing the ratio profile among different locationsalong the reference metaphase chromosomes, said ratio profile at eachlocation being proportional to the ratio of the copy number of the DNAsequence that bind to that location in the first subject cell or cellpopulation to the copy number of substantially identical sequences inthe second cell or cell population.

Said representative methods can further comprise comparing copy numbersof different DNA sequences in more than two subject DNAs wherein thecomparing is done pairwise between the signals from each subject DNA.

This invention further discloses methods to determine the ratio of copynumbers of different DNA sequences in one subject cell or cellpopulation to copy numbers of substantially identical sequences inanother cell or cell population wherein the steps of (a) through (f) asdescribed above are performed as well as the following steps:

g. determining the average copy number of a calibration sequence in bothsubject cells or cell populations, said calibration sequence beingsubstantially identical to a single copy sequence in the referencemetaphase cells; and

h. normalizing the ratio profile calculated in (f) so that at thecalibration position, the ratio profile is equal to the ratio of theaverage copy numbers determined in (g), the normalized ratio profile atany other location along the reference metaphase chromosomes therebygiving the ratio of the copy numbers of the DNA sequences in the twosubject DNAs that bind at that location. That method can be extended tofurther subject nucleic acids as for example determining the ratio ofcopy numbers of DNA sequences in more than two subject DNAs wherein thecomparing is done pairwise between signals from each subject DNA.

Further disclosed are methods for comparing copy numbers of differentDNA sequences in a test cell or cell population, said method comprisingapplying steps (a) through (e) of the above-described methods and

f. observing and/or measuring the intensities of the signal from eachsubject DNA, and the relative intensities, as a function of positionalong the reference metaphase chromosomes wherein one of the subjectcells or cell populations is the test cell or cell population and theother is a normal cell or cell population; and

(g) comparing the relative intensities among different locations alongthe reference metaphase chromosomes, wherein the greater the relativeintensity at a location, the greater the copy number of the sequence inthe test cell or cell population that binds to that location, except forsex chromosomes where the comparison needs to take into account thedifferences in copy numbers of sequences in the sex chromosomes inrelation to those on the autosomes in the normal subject cell or cellpopulation.

A related representative method is that for comparing the copy number ofdifferent DNA sequences in a test cell or cell population comprisingapplying steps (a) through (e) of the above described methods whereinone of the subject cells or cell populations is the test cell or cellpopulation, and the other is a standard cell or cell population whereinthe copy numbers of the DNA sequences that bind to different positionson the reference metaphase chromosomes is known and steps:

f. measuring the intensities of the signals from each of the boundsubject DNAs and calculating the ratio of intensities as a function ofposition along the reference metaphase chromosomes to form a ratioprofile;

g. adjusting the ratio profile at each location along the referencemetaphase chromosomes by multiplying the ratio profile by the known copynumber of DNA sequences in the standard cell or cell population thatbind there; and

h. comparing the adjusted ratio profiles at different locations alongthe reference metaphase chromosomes wherein the greater the adjustedratio profile at a location, the greater the copy number of the DNAsequence in the test cell or cell population that binds there.

Another related representative method is that for determining the ratiosof the copy numbers of different DNA sequences in a test cell or cellpopulation, said method comprising applying steps (a) through (f) of,theimmediately above-described method and the steps of adjusting the ratioprofile at each location along the reference metaphase chromosomes bymultiplying the ratio profile by the known copy number of sequences thatbind there; and calculating the ratio of the copy number of a DNAsequence in the test cell or cell population that binds to one locationon the reference metaphase chromosomes to the copy number of a sequencethat binds to another location by dividing the adjusted ratio profile atthe location of the first sequence by that at the location of thesecond. Said representative method can be extended to determine the copynumber of different DNA sequences in a test cell or cell populationwherein steps (a) through (f) as described above are followed and thenthe following steps of adjusting the ratio profile at each locationalong the reference metaphase chromosomes by multiplying the ratioprofile by the known copy number of DNA sequences in the standard cellor cell population that bind there;

determining the copy number of a calibration sequence in the test cellor cell population that is substantially identical to a single copysequence in the reference cells; and

normalizing the adjusted ratio profile so that at the location of thecalibration sequence on the reference metaphase chromosomes, thenormalized, adjusted ratio profile is equal to the copy number of thecalibration sequence determined in the above step, the value of thenormalized, adjusted ratio profile at another location then being equalto the copy number of the DNA sequence in the test cell or cellpopulation that binds at that location. That method can be analogouslyperformed wherein two or more calibration sequences are used, and theadjusted ratio profile is normalized to get the best fit to the copynumbers of the ensemble of calibration sequences. Preferably, the copynumber of the calibration sequence is determined by in situhybridization. Those methods can comprise in situ hybridizing probes formore than one calibration position and normalizing to obtain the bestfit of the ratio profile to the calibration positions. The standard cellor cell population preferably have normal genomes. In many applicationsof CGH, the reference metaphase chromosomes are normal.

Further, this invention concerns the use of antenna cell lines Anexemplary method is for detecting amplification of a certain sequence orgroup of sequences in a subject cell or cell population, comprisingessentially steps (a) through (e) of the above-described methods whereinthe in situ hybridization is targeted to antenna cells in which the DNAsequences to be tested for is or are amplified, and examining thereference cell for regions that are hybridized significantly moreintensely than others, the presence of such regions indicatingamplifications of the sequences which are being tested. The chromosomesof said antenna cell lines may be in interphase or in metaphase.

When a single labeled subject nucleic acid is being hybridized, or ifmultiple labeled subject nucleic acids are hybridized sequentially, itis important that the binding sites on the reference genome not besaturated prior to observing and/or measuring the signal intensity(ies).In the case of a single labeled subject nucleic acid, nonsaturation canbe effected in a number of ways, for example, by stopping thehybridization, by providing insufficient subject nucleic acid, and/or byproviding a sufficient amount of unlabeled nucleic acid which issufficiently complementary to the reference chromosomes to competitivelyprevent saturation of sites therein by the labeled subject nucleic acid.

When there are two or more labeled subject nucleic acids, those subjectnucleic acids can be hybridized in situ to the reference genomesequentially or simultaneously. simultaneous in situ hybridization ispreferred in that saturation of the targeted binding sites in thereference genome will not interfere with the procedure. When sequentialin situ hybridization is used, it must be performed under conditionswherein the individual hybridizations are stopped well before thebinding sites on the reference chromosomes are saturated.

Objects of this invention are to detect sequence copy number imbalancesthroughout an entire genome in one hybridization, to map gains and/orlosses of sequences in a genome, and/or to provide a copy numberkaryotype of a subject genome.

Further, an object of this invention is to enable the detection ofrelative copy number differences that are common to a number ofdifferent cells and/or cell populations. For example, CGH methods can beused wherein DNAs extracted from cells of many different tumors arecombined and labeled; the hybridization of those combined labeled DNAsto normal condensed chromosomes, provides for the rapid identificationof only those copy number changes that occurred in mos: of the tumors.Less frequently occurring variations would be averaged out. Thus, thisinvention further provides for a CGH method wherein two or more of thesubject nucleic acids that were extracted from different cells and/orfrom numbers of cells from different cell populations, are labeled thesame, and hybridized to a reference spread under conditions whereinrepetitive sequences are removed and/or suppressed and wherein sequencecopy number differences that are common in said combined labeled nucleicacid sequences are determined.

Another object of this invention is to provide the means ofcytogenetically analysing archived chromosomal material, that is, fixedmaterial from, for example, biopsied tissue specimens, preferablycataloged and keyed to medical records of patients from whom thespecimens were taken, and archaeological chromosomal material. Suchchromosomal material cannot, of course, be karyotyped according totraditional means in that no live cells are present to culture and fromwhich to prepare chromosomal spreads. However, the nucleic acid can beextracted therefrom and amplified by a polymerase chain reaction (PCR)procedure or by a non-PCR procedure and tested by the methods of thisinvention.

This invention further provides for a method to detect simultaneously anensemble of amplifications and/or deletions in a tumor wherein theresults can be used to determine the subsequent behavior of that tumor.Said determination is made by associating the patterns of amplificationsand/or deletions in tumor cells with the behavior of that tumor. Suchassociations can be made by testing, for example, as indicatedimmediately above, DNA from archived tumor tissue keyed to medicalrecords, or when fresh tumor specimens are tested by CGH and thepatients are followed. Further, such associations can be made with CGHmethods wherein there are more than one subject cell and/or cellpopulation, for example, one or more tumors.

Another object of this invention is to provide a method of analyzingcells from a suspected lesion at an early stage of development. Anadvantage of the methods of this invention is that only a few cells arenecessary for the analysis. The early detection of amplifications and/ordeletions in cells from a lesion allow for early therapeuticintervention that can be tailored to the extent of, for example,invasiveness known to be associated with such genetic rearrangements.Further, such early detection provides a means to associate theprogression of the cells with the genetic rearrangements thereindetected by the methods of this invention.

Tumors can be karyotypically heterogeneous containing therein variouspopulations of cells each having different types of geneticrearrangements. As indicated above tumor cells are difficult to culture,and it is not clear that cultured cells are representative of theoriginal tumor cell population. This invention provides the means toby-pass the culturing obstacle and allows genetic characterization oftumor cells and thus, of the heterogeneity of tumors by testing cellsfrom different subregions thereof according to the methods of thisinvention. Bulk extraction of the nucleic acid from many cells of atumor can also be used to test for consistent amplifications and/ordeletions within a tumor.

It is another object of this invention to provide methods of detectingamplifications and/or deletions of nucleic acid sequences whereincertain cell lines termed herein "antenna cell lines", are used toenhance the sensitivity of the detection.

It is still further an object of this invention to provide methods ofprenatal or perinatal analysis wherein the nucleic acid of the child'scells is extracted and tested according to the methods of thisinvention. In one embodiment of CGH, such material is human andhybridized to a normal human metaphase spread to detect whether anydeletions and/or amplifications are therein present, for example, anextra copy of chromosome 21, diagnostic for Down syndrome. Test kits forperforming CGH methods are also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the results of a CGH hybridization of DNA from theBT-474 human breast cancer cell line to a metaphase spread of normalperipheral bloodlymphocyte human chromosomes. The BT-474 cell line isknown to have a 13-fold c-erbB-2 amplification. The DNA from that cellline was labeled with digoxigenin-11-dUTP and stained with fluoresceinisothiocyanate (FITC); signals from the hybridization of the cell lineDNA are green in the photomicrograph. A chromosome 17 peri-centromericrepeat probe (cosmid cK 17.10) was labeled with biotin-14-dATP andstained with Texas Red; signals from that probe's hybridization are red.The chromosomal DNA was counterstained with 4,6-diamidino-2-phenylindole(DAPI) resulting in a blue counterstaining. The photomicrograph wastaken using a multicolor image analysis system after contrast stretchingand pseudocolor display.

The green signals indicating amplified sequences in the BT474 cell lineare seen in FIG. 1 at the following loci: 17q12 (the erbB-2 locus),17q22-q23 and 20q13-ter. The latter two sites were previouslyunrecognized sites of amplification in that cell line. One centromericrepeat is non-specifically stained green.

FIG. 2 schematically illustrates the general approach used in performingthe methods of this invention-Comparative Genomic Hybridization (CGH).The reference chromosome spread is hybridized with various nucleic acidmixtures, either simultaneously or at different times, to obtain thedesired information. Representative mixtures could include unlabeledsequences designed to block sequences in the various other nucleic acidpools, for example, the high-copy repetitive sequences in human genomicDNA; unlabeled competitor nucleic acid to prevent saturation of thetarget sites for the labeled mixtures, for example, human genomic DNAwithin a factor of 10 of the concentration used for the labeled subjectnucleic acids (see FIG. 5); and one or more pools of sequences ofdifferent origin that are differently labeled so that their binding canbe independently assessed, for example, tumor and normal genomic DNA(see FIGS. 6 and 7). The information on the sequence frequency of thelabeled pools is obtained by analysis of the intensity of the individualsignals and/or the differences in ratios of intensities among thesignals as a function of position along the reference chromosomes.

FIG. 3 outlines general aspects of the CGH procedure used in Example 1,infra. The reference chromosome spread, in this example normal humanchromosomes, is first hybridized for about one hour with a highconcentration of unlabeled human genomic DNA (FIG. 3a). Thatprehybridization blocks many of the high copy repetitive sequences inthe chromosomes so that the high copy repetitive sequences in thelabeled subject nucleic acid, in this case labeled tumor DNA, will notsubstantially contribute to the signal during the subsequenthybridization. The labeled tumor DNA, and perhaps some competitor DNA orother comparison nucleic acid are then hybridized to the targetreference spread (FIG. 3b). Cot1 DNA can be included, in thehybridization as in Example 1, below to block more effectively thecentromeric repetitive sequences in the labeled subject nucleic acids.

FIG. 3 is representative of one way of reducing signals from repetitivesequences. Other methods are detailed herein infra. In each of the CGHmethods including the procedures outlined in the rest of the figures,some means of reducing the signal from the repetitive sequences is used,but not specifically indicated in the figures. It is important for CGHthat the signal from each subject nucleic acid be dominated by sequencesthat bind to well defined loci. Total suppression of the signal from thegenomic repeats is not necessary, but the poorer the suppression, theless able the procedure is to detect small differences in sequencefrequency.

FIG. 4 illustrates the procedure used in Example 1, for whichrepresentative results are shown in the photomicrographs of FIGS. 1 and8. As shown in FIG. 4a, labeled human tumor DNA is hybridized to anormal human chromosome spread. [Please note as indicated in thedescription for FIG. 3, provisions were made to suppress the signal fromthe repetitive sequences although those provisions are not specificallyindicated in the figure. Example 1 details one preferred method tosuppress the hybridization signals from repetitive sequences.] In thisrepresentative example, the tumor DNA is assumed to contain a regionwherein some sequences are highly amplified, for example, an ampliconcontaining an oncogene. The amplified sequences in the tumor DNA may beclustered and integrated in some tumor chromosomes; they may beintegrated into multiple places in the tumor genome; or, they may existas extra-chromosomal elements. The sequences of the amplicon will map tosome chromosomal location in the reference genome, which in this case isa normal human genome.

The lower portion of FIG. 4 illustrates the kinetics of the build-up ofthe signal on a target reference chromosome. The signal builds morerapidly in the amplified region since more copies of those sequences areavailable for hybridization. If the reaction is stopped before thetarget chromosome is saturated, or if insufficient labeled S DNA isadded to achieve saturation, then the genomic region that was amplifiedin the tumor will appear higher in intensity on the normal chromosome asillustrated by the dark band on the left reference chromosome. The moreintensely labeled region (dark band) indicates the location and extentof the amplicon as reflected in the reference genome. Thus, theamplification is detected without prior knowledge of its existence, andthe origin of the amplified sequences is mapped in the normal humangenome.

If the reaction illustrated in FIG. 4 is allowed to proceed tosaturation of the target sites, contrast is lost, as shown by therepresentative reference chromosome on the right. Thus, in thisembodiment of CGH, it is important to stop the hybridization beforesaturation of the target or provide insufficient probe for saturation.The graphs schematically show the build-up of the hybridization signalin the region that was amplified (graph on right) and in the remainderthat was unamplified (graph on left). The arrows connect the chromosomalregions with the times of observation on the kinetic curve.

FIG. 5 illustrates an embodiment of CGH that avoids the potentialsaturation of the target as shown in the lower right portion of FIG. 4.In this representative example, the reference nucleic acid is a humanchromosome spread; the subject nucleic acid is labeled tumor DNA. Ifunlabeled human genomic DNA is included with the labeled tumor DNA inexcess, in this case at a five-fold higher concentration than that ofthe labeled tumor DNA, then any saturation of the target will be due toa combination of labeled and unlabeled copies of the nucleic acidsequences, rather than just labeled copies as shown in the lower rightof FIG. 4. [Once again, as indicated in FIGS. 3 and 4 the means ofreducing the signal from repetitive sequences is not indicated in thisfigure, but it is assumed that some protocol is performed to removesubstantially the repetitive sequences that would bind to multiple lociin the reference genome and/or to block such sequences from binding tothe target.]

At the early stages of the reaction, the amplified region will build upfaster than elsewhere in the chromosome (for example if the sequence isamplified five-fold, it would build up 5 times as fast) and will bedetectable as in the lower left of FIG. 4. However as the reactionproceeds to saturation, the unamplified regions of the chromosome reachonly one-fifth (1/5) of the intensity shown in FIG. 4, because most ofthe sites are filled by unlabeled copies of the sequences. On the otherhand, a sequence that was amplified five-fold in the tumor would reachone-half (1/2) of the saturation intensity since an equal number oflabeled and unlabeled copies of those sequences are present. Thus,contrast is maintained according to this embodiment at all stages of thereaction, although it changes as the reaction proceeds.

FIG. 6 illustrates an embodiment of CGH designed to enhance itssensitivity in detecting small changes in copy number of varioussequences. When a CGH procedure as indicated in FIG. 5 is followed,intrinsic variation in the saturation levels, or rate of signal build-upat different positions in the reference genome may not be indicative ofabnormal gain or loss of sequences. Such intrinsic variations wouldinterfere with interpretation of intensity differences as indicatingdifferences in copy number of the sequences. This CGH embodimentovercomes that potential problem by providing a mixture of labeledsubject nucleic acid, in this case tumor DNA labeled with a greenfluorochrome, and a differently labeled competitor nucleic acid in thiscase normal human genomic DNA labeled with a red fluorochrome. The twvodifferently labeled DNAs are simultaneously hybridized to the chromosomespread. [Once again, removal of the repetitive sequences and/or blockingof the signal therefrom is performed but not illustrated.] Changes inthe ratio of green to red along each of the chromosomes in the referencespread then indicate regions of increased or decreased sequence copynumber in the tumor. Those ratio changes may result in color variationsfrom red to yellow to green on the reference spread.

FIG. 7 graphically and schematically explains the kinetics underlyingthe CGH embodiment illustrated in FIG. 6. In the center is one of thechromosomes of the reference chromosome spread, a normal humanchromosome in this care. The darkness of the shading on the referencechromosome shows the ratio of green to red intensity along thechromosome.

In the amplified region, the green/red ratio is much higher than in thenormal region, whereas in the deleted region the green/red ratio is lessthan in the normal region. The arrows from examples of each of thedifferent green/red intensity regions point to kinetic curves thatindicate the build-up of green (solid line for the tumor DNA) and red(dashed line for the normal DNA) signals during the hybridization. Inthe normal region, upper left graph, the red and green signals buildtogether. (They have been normalized to be equal for the purposes ofthis explanation.) In the amplified region, upper right, the green(tutor) signal builds up much more rapidly than the red (normal) signal,the green/red ratio being approximately the level of amplification(given the normalization to the normal part of the chromosome).

In the lower left of FIG. 7, the signal build-up for the duplicatedregion is shown; the green (tumor) signal is 50% brighter than the red(normal) signal. In the lower right, the build-up for a deleted regionis schematically described; the green (tumor) signal is 50% dimmer thanthe red (normal) signal. The ratio approach of this CGH embodimentfurther normalizes for the frequent finding that hybridization to somechromosomes in a spread is intrinsically brighter than that for othersbecause of differences in the local hybridization environment.

FIG. 8 illustrates an example of how a deletion can be detected usingCGH. A deletion is simulated by employing DNA from a human primarybreast carcinoma (XX) as a subject genome and a normal male chromosomespread (XY) as the reference genome. The absence of the Y-chromosome inthe tumor DNA was detected, as would a cytogenetically significantdeletion, by the hybridization. DNA from the primary breast carcinomawas labeled with digoxigenin-11DUTP and stained with fluoresceinisothiocyanate (FITC) (green signals). The normal male peripheral bloodlymphocyte metaphase was counterstained with 4,6diamidino2-phenylindole(DAPI) (blue). The picture was taken from a multicolor image analysissystem (QUIPS) after image thresholding and contrast stretching. Thegreen chromosomal fluorescence level on all chromosomes was increased tomake the absence of this fluorescence on the Y-chromosome (arrow) morereadily visible. The Y-chromosome is only stained with the DAPIcounterstain.

FIG. 9 presents an idiogram of chromosome 1 from the breast cancer cellline 600 MPE, the karyotype for which was published by Smith et al.,JNCI, 78: 611-615 (1987).

FIG. 10A is a photomicrograph showing the comparative genomichybridization (CGH) of DNA from a 45, XO cell line (green) and a normalhuman female DNA (red) to a normal human male reference spread. Thereddish color of the X chromosome, pointed out by the large arrow, ascompared with the autosomes reflects the lower relative copy number ofthe X chromosome sequences in the 45, XO cell line. Faint staining of asmall part of the Y chromosome, pointed out by the small arrow, is aresult of the binding of homologous sequences in the pseudo-autosomalregion.

FIG. 10B graphically illustrates the correlation of the number of Xchromosomes in five fibroblast cell lines and the average green-to-redratio of the X chromosome(s) relative to the same ratio for theautosomes.

FIG. 11 illustrates green-to-red fluorescence ratio profiles ofchromosomes 1, 9, 11, 16 and 17 after comparative genomic hybridizationwith breast cancer cell line 600PE (green) and with a normal DNA (red).The profiles reflect the relative copy number of the chromosomalregions. Fluorescence in situ hybridization (FISH) with 16p and 16qcosmid probes to interphase and metaphase 600PE cells indicated thatthere were two signals with 16p cosmid probes and one signal from the16q cosmid probes. That information on the absolute copy number of thoseloci provided by FISH permits interpretation of the ratio 1.0 asindicating that there are two copies of the sequence throughout thegenome.

The dip in the profile at 1p34 through 1p36 may represent a previouslyunsuspected small interstitial deletion; however, that observation hasnot yet been independently verified with specific probes for thatregion.

Centromeric and heterochromatic regions of the genome are not includedin the analysis because the Cot-1 DNA partially blocks signals in thoseregions, and the large copy number polymorphisms between individualsequences at those loci effect unreliable ratio data.

FIG. 12(A) and 12(B) respectively provide green-to-red fluorescenceratio profiles of chromosome 8 (A) and chromosome 2 (B) aftercomparative genomic hybridization respectively with COLO 320 HSR (humancolon adenocarcinoma cell line) and NCI H69 (small cell lung carcinomacell line) cell line DNAs (green) and with normal human DNA (red). Theinserts illustrate the overlaid green and red fluorescence images of thechromosomes, and the chromosomal medial axis drawn by the image analysisprogram used.

In FIG. 12(A), the myc locus at 8q24 shows a highly elevatedgreen-to-red ratio, which is consistent with the known high levelamplification of myc in the COLO 320HSR cell line.

In FIG. 12(B), three regions of amplification are seen on chromosome 2.The signal at 2p24 corresponds to the location of N-myc known to beamplified in the NCI-H69 cell line. The two other regions with a highlyincreased green-to-red fluorescence ratio, at 2p21 and 2q21, were notpreviously known to be amplified in the NCI-H69 cell line.

FIG. 13 is a photomicrograph of a comparative genomic hybridization(CGH) with BT-20 (breast cancer cell line) cell line DNA (green) andnormal DNA (red) to a normal human metaphase spread. Loss of DNAsequences in the tumor cell line DNA relative to normal DNA are shown byred whereas gain of DNA sequences in the tumor cell line are shown ingreen.

FIG. 14. Quantitation of green to red fluorescence intensities along thetwo homologues of chromosomes 1, 8, 10, 16 and 20 after CGH with primarybreast cancer DNA in green and normal DNA in red. An increased ratio isobserved at 8q (high-level gain) and at lq (low-level gain). Regionalcopy number increases at 10q22 and at 20q12-q13 are also evident,whereas chromosome 16 shows no changes. The ratios are normalized sothat the average green to red ratio for each metaphase cell is 1.00.Ratio changes at repeat-rich heterochromatic regions are not reliableand are displayed with dotted lines. The chromosome diagrams below eachratio profile are shown only for approximate visual comparison and werenot used for localizing the changes.

FIG. 15. Chromosomal localization of DNA sequence copy number increasesin 33 primary breast tumors (left side of the chromosome diagrams) and15 breast cancer cell lines (right). Low-level copy number increases areshown in blue and high-level in red. The chromosomal band location ofthe changes was determined directly based on the DAPI banding for eachchromosome. Because high-resolution sub-band localization was notpossible using DAPI staining, the regions shown in the figure are oftenlarger than the actual size of the amplicon.

FIG. 16 shows the gains and losses of DNA sequences in primary bladdercarcinomas.

FIG. 17 summarizes the CGH and LOH data for ovarian cancers bychromosome arm in FIG. 16 for 3C Grade III tumors.

FIG. 18 shows gene dosage abnormality detected by CGHE analyses of 30grade III ovarian cancers.

FIG. 19 shows a schematic illustration of a model of progression inwhich tumor progress as a result of accumulation of genetic aberrations,some of which confer the same phenotype.

FIG. 20 shows: Panel a. chromosomal locations of cosmid probes mappedalong chromosome 20. Panel b. Level of amplification BT474 alongchromosome 20 in interphase nuclei determined using FISH with mappedprobes.

DETATLED DESCRIPTION

Comparative Genomic Hybridization (CGH) has also been termed Copy RatioReverse Cytogenetics (CRRC), competition hybridization and quantitativein situ ratio karyotyping (QUIRK). Further, in the embodiment whereinfluorochromes are used as labels, it has been termed competition FISH(fluorescence in situ hybridization). CGH specifically provides methodswhereby amplifications, duplications and/or deletions can be identifiedin an immediate overview of a genome.

CGH provides methods for determining variations in the copy number ofdifferent elements in a mixture of nucleic acid sequences (for example,genomic DNA isolated from a tumor) as a function of the location ofthose sequences in the genome of a reference organism (for example, thegenome of a normal cell from the same species). The methods comprise theuse of in situ hybridization of the nucleic acid sequence mixture to achromosome spread of the reference organism, and measuring the intensityof the hybridization at different locations along the targetchromosomes. Exemplary methods are schematically outlined in FIGS. 2-7.Those illustrative examples are not exhaustive but suggest the widerange of variations and other uses of the basic approach.

As the figure descriptions indicate, it is critical that signals fromrepetitive sequences do not dominate the signal from the subject nucleicacid pool, and that they be removed from the pool or that their signalsbe suppressed as necessary. It is preferred to exclude sequences fromthe hybridization or block sequences in the hybridization mixture thatcould bind to multiple clearly separated positions on the chromosomes,for example, sites that are on different chromosomes, or that are on thesame chromosome but are well-separated. In many applications of CGH, itis the high copy repetitive sequences, such as Alu, Kpn, Lines, andalpha-satellites among others, that are removed from the labeled subjectnucleic acid and/or which are blocked and/or the binding sites thereforare blocked. Described herein are methods to remove and/or block thoserepetitive signals. It should be noted that nucleic acid sequences inthe labeled nucleic acid that bind to single copy loci are substantiallyretained in the hybridization mixture of labeled subject nucleic acids,and such single copy sequences as well as their binding sites in thereference chromosome spread remain substantially unblocked relative tothe repetitive sequences that bind to multiple loci (that is, loci thatare visually distinguishable) both before and during the hybridization.

The methods of this invention provide the means to identify previouslyunknown regions of amplification and deletion. For example, oneembodiment of CGH as detailed in Example 1 herein provides an efficientmethod that gives an immediate overview of a genome identifying allregions that are amplified greater than about five-fold to ten-fold aswell as at least large deletions. More sensitive embodiments that canidentify smaller amplifications and deletions are also disclosed.

Nanogram quantities of the subject nucleic acids are required for theCGH methods of this invention. Paraffin embedded tumor sections can beused as well as fresh or frozen material. Snap frozen material fromnormal and malignant tissue are preferred for mRNA isolation.

Standard procedures can be used to isolate the required nucleic acidfrom the subject cells. However, if the nucleic acid, for example, DNAor mRNA, is to be extracted from a low number of cells (as from aparticular tumor subregion) or from a single cell, it is necessary toamplify that nucleic acid, by a polymerase chain reaction (PCR)procedure or by a non-polymerase chain reaction (non-PCR) procedure. PCRand preferred PCR procedures are described infra. Exemplary non-PCRprocedures include the ligase chain reaction (LCR) and linearamplification by use of appropriate primers and their extension (randompriming).

Some of the various embodiments of CGH are illustrated, particularly inFIGS. 2-7. In the embodiment illustrated in FIGS. 6 and 7, wherein asubject nucleic acid, in this case, human genomic DNA, that is labeleddifferently from another subject nucleic acid, amplifications and/ordeletions are indicated by a change in ratio between the differentsignals, rather than just a change in signal intensity.

The representative examples concerning CGH of Examples 1, 2 and 3 belowinvolve the hybridizations of tumor cell line DNA to normal humanmetaphase spreads. However, there are many permutations and combinationsof pairwise and multiple hybridizations of different nucleic acids fromdifferent genomes all of which are considered to be within the scope ofthis invention.

For example, CGH could be used to hybridize labeled DNA from a tumorcell line to metaphase spreads of that same cell line to estimate thelevel and pattern of amplification in each cell line, comparing thoseresults to hybridizations of said tumor cell line DNA to a normal humanmetaphase spread. Alternatively, labeled tumor cell line DNA anddifferently labeled human genomic DNA could be simultaneously hybridizedto a metaphase spread of a tumor cell line metaphase spread. Further,DNA from a primary tumor and that from its metastasis could bedifferently labeled and hybridized in a CGH method to a normal humanmetaphase or to a related tumor cell line metaphase. Those are just someof the many examples of CGH.

Although the examples herein concern the hybridizations of the DNA frombreast cancer cell lines and primary tumors to normal human metaphasespreads, it will be clear to anyone skilled in the art that CGH is notlimited to studying genomes of cancer cells or to the results ofhybridizing abnormal genomes to normal genomes. CGH permits thecomparison of nucleic acid sequence copy frequencies of any two or moregenomes, even genomes of different species if their nucleic acidsequences are sufficiently complementary to allow for meaningfulinterpretation. It should be noted regarding interspecies comparisonsthat the information obtained by CGH includes not only an assessment ofrelative copy number but also that of sequence divergence.

It will also be clear to those skilled in the art that hybridizationwith nucleic acid other than chromosomal DNA, such as messenger RNA(mRNA) or complementary DNA (cDNA) of subject cells can be used todetermine the location and level of expression of genes in those cells.Conventional methodology is used to extract mRNA from a cell or cellpopulation, and to synthesize in vitro c-DNA by reverse transcription.

CGH does not require the preparation of condensed chromosomes, forexample, metaphase, prophase or other condensed chromosomal states, ofthe subject genomes. Thus, genomes from which metaphase, prophase orotherwise condensed chromosomal spreads are difficult, time-consuming ornot possible to prepare at least in good quality, for example, genomesof tumor cells or fetal cells can be studied by CGH.

In CGH, labeled subject nucleic acids, for example, labeled tumor DNA,is hybridized to a reference genome, for example, a normal humanmetaphase spread, under conditions in which the signal from amplified,duplicated and/or deleted nucleic acid sequences from the labelednucleic acid can be visualized with good contrast. Such visualization isaccomplished by suppressing the hybridization of repetitive sequencesthat bind to multiple loci including the high copy interspersed andclustered repetitive sequences, such as, Alu, Kpn, Lines,alphasatellites among others, using unlabeled total human genomicnucleic acid, preferably DNA, and/or the repeat-enriched (Cot-1)fraction of genomic DNA, and/or by removing such repetitive sequencesfrom the hybridization mixture. In providing the detection sensitivityrequired, the extent of suppression of the hybridization of repetitivesequences and/or removal thereof can be adjusted to the extent necessaryto provide adequate contrast to detect the differences in copy numberbeing sought; for example, subtler copy number changes may require thesuppression or removal of lower level repetitive sequences.

When combining more than one labeled nucleic acid in a hybridizationmixture, the relative concentrations and/or labeling densities may beadjusted for various purposes. For example, when using visualobservation or photography of the results, the individual colorintensities need to be adjusted for optimum observability of changes intheir relative intensities. Adjustments can also be made by selectingappropriate detection reagents (avidin, antibodies and the like), or bythe design of the microscope filters among other parameters. When usingquantitative image analysis, mathematical normalization can be used tocompensate for general differences in the staining intensities ofdifferent colors.

The kinetics of the CGH hybridizations are complicated. Since thesubject nucleic acids are frequently double stranded, complementarysequences will reassociate in the hybridization mix as well ashybridizing to the target. Such reassociation may result in a more rapiddecrease in concentration of the high copy sequences than the low copyones, thereby maklng the signal intensity variations on the referencechromosomes less pronounced than the copy differences in the originalsubject DNAs. In addition, non-specific binding of the labeled subjectDNAs to the slide, coverslip, etc., may generally reduce theconcentration of that labeled subject nucleic acid during thehybridization. Those skilled in the art will recognize numerous methodsof optimizing the quantitative aspects of CGH, such as, mathematicalcorrection of digital images, supplying freshly denatured subject DNAduring the hybridization, and adding unlabeled genomic DNA in excess todominate the reassociation rates.

The resolution of CGH is presently at a level that can be seen through alight microscope, as is traditional cytogenetic staining. Thus, if asmall sequence in a subject nucleic acid is amplified, to be seen as asignal in a subject genome, it must be amplified enough times for itssignal to be able to be visualized under a light microscope. Forexample, the locus for erbB-2 which is relatively small (veryapproximately, a few hundred kb), needs to be amplified at least greaterthan five times to be visually distinguishable under a light microscopewhen the CGH embodiment used in Example 1 is employed. On the otherhand, if a large section of a chromosome is present at increasedfrequency in a subject nucleic acid, the signal from that region wouldshow up in the reference genome at a much lower level of amplification.

The term "labeled" is herein used to indicate that there is some methodto visualize nucleic acid fragments that are bound to the target,whether or not the fragments directly carry some modified constituent. Asection infra entitled "Labeling the Nucleic Acid Fragments of theSubject Nucleic Acids" describes various means of directly labeling theprobe and other labeling means by which the bound probe can be detected.

The phrase "antenna cell line" is herein used to indicate a referencegenome that has one or more known significant genetic aberrations, forexample, a cell line known to have an oncogene that is highly amplified,for example, in large homogeneously staining regions (HSRs). Theamplified regions of that cell line would thus provide a much biggertarget site than a normal chromosome spread. Thus, observation of thesignal from such a large target site would be easier in that on averagethe signal would be brighter from amplified target sequences in thereference genome as provided by such an antenna cell line. A subjectnucleic acid extracted from, for example, a number of tumor cells, couldbe tested by a CGH hybridization to such an antenna cell line to see ifit also contained amplifications of the oncogene known to be amplifiedin the cell line.

When an antenna cell line is used as the reference genome, there areinstances wherein it can be used in interphase rather than as achromosome spread. For example, if one is checking to see if a certainoncogene is amplified or not in the subject nucleic acid, interphase CGHis sufficient. However, the maximum amount of information is providedwhen condensed chromosome spreads are used.

A base sequence at any point in the genome can be classified as either"single-copy" or "repetitive". For practical purposes the sequence needsto be long enough so that a complementary probe sequence can form astable hybrid with the target sequence under the hybridizationconditions being used. Such a length is typically in the range ofseveral tens to hundreds of nucleotides.

A "single-copy sequence" is that wherein only one copy of the targetnucleic acid sequence is present in the haploid genome. "Single-copysequences" are also known in the art as "unique sequences". A probecomplementary to a single-copy sequence has one binding site in haploidgenome. A "repetitive sequence" is that wherein there is more than onecopy of the same target nucleic acid sequence in the genome. Each copyof a repetitive sequence need not be identical to all the others. Theimportant feature is that the sequence be sufficiently similar to theother members of the family of repetitive sequences such that under thehybridization conditions being used, the same fragment of probe nucleicacid is capable of forming stable hybrids with each copy.

Herein, the terms repetitive sequences, repeated sequences and repeatsare used interchangeably.

The phrase "metaphase chromosomes" in herein defined to encompass theconcept of "condensed chromosomes" and is defined to mean not onlychromosomes condensed in the prophase or metaphase stage of mitosis butany condensed chromosomes, for example, those condensed by prematurechromosome condensation or at any stage in the cell cycle wherein thechromosome can be visualized as an individual entity. It is preferredthat the chromosomes in the reference genome be as long as possible butcondensed sufficiently to be visualized individually.

A subject nucleic acid is herein considered to be the same as anothernucleic acid if it is from a member of the same sex of the same speciesand has no significant cytogenetic differences from the other nucleicacid. For example, the DNA extracted from normal lymphocytes of a humanfemale is considered for the purposes of this invention to be the samenucleic acid as that of DNA from normal cells of a human femaleplacenta.

The following abbreviations are used herein:

ABBREVIANTS

AAF--N-acetoxy-N-2-acetyl-aminofluorene

ATCC--American Type Culture Collection

BN--bicarbonate buffer with NP40

BRL--Bethesda Research Laboratories

bp--base pair

CCD--charge coupled device

CGH--Comparative Genomic Hybridization

Chr.--chromosomal

CML--chronic myelogenous leukemia

CRRC--Copy Ratio Reverse Cytogenetics

DAPI--4,6diamidino-2-phenylindole

dATP--deoxyadenosine triphosphate

DCS--as in fluorescein-avidin DCS (a commercially available cell sortergrade of fluorescein Avidin D)

DCIP--deoxycytosine triphosphate

DGIT--deoxyguanosine triphosphate

DI--DNA index

DM--double minute chromosome

DNTP--deoxynucleotide triphosphate

DTP--deoxythymidine triphosphate

DUTP--deoxyuridine triphosphate

EDTA--ethylenediaminetetraacetate

E/P--estrogen/progesterone

FISH--fluorescence in situ hybridization

FACS--fluorescence-activated cell sorting

FITC--fluorescein isothiocyanate

HPLC--high performance liquid chromatography

HSR--homogeneously staining region

ISCN--International System for Cytogenetic Nomenclature

IB--isolation buffer

kb--kilobase

kDa--kilodalton

LOH--loss of heterozygosity

Mb--megabase

met.--metastasis

min--minute

ml--milliliter

mm--millimole

mm--millimeter

ng--nanogram

NIGMS--National Institute of General Medical Sciences

NP40--non-ionic detergent commercially available from Sigma as NonidetP40 (St. Louis, Mo.)

PBS--phosphate-buffered saline

PCR--polymerase chain reaction

PHA--phytohemagglutinin

Pi--propidium iodide

Pl.--pleural

PMSF--phenylmethylsulfonyl fluoride

PN--mixture of 0.1 M NaH₂ PO₄ and 0.1 M buffer Na₂ HPO₄, pH 8; 0.1%NP-40

PNM--Pn buffer plus 5% nonfat dry milk buffer (centrifuged); 0.02% Naazide

QUIRK--quan titative in situ ratio karyotyping

Rb-1--retinoblastoma tumor suppressor gene

RFLP--restriction fragment length polymorphism

RPM--revolutions per minute

SD--Standard Deviation

SDS--sodium dodecyl sulfate

ssc 0.15 M NaCl/0.015 M Na citrate, pH 7

Td--edoubling time

ug--microgram

ul--microliter

um--micrometer

um--micromole

VNTR--variable number tandem repeat

Resolution of differences in copy number can be improved by the use ofimage analysis and by averaging the results from hybridizations of asubject nucleic acid to multiple condensed chromosome spreads. Usingsuch methods, the background signal (noise) can be differentiated fromactual nucleic acid sequence copy number differences.

Image-Analysis:

An image analysis system, preferably computer assisted, can be used toenhance and/or accurately quantitate the intensity differences betweenand/or among the signals from a hybridization and the backgroundstaining differences for more accurate and easier interpretation ofresults. Image analysis and methods to measure intensity are described,for example, in Hiraoka et al., Science, 238: 36-41 (1987) and Aikens etal., Meth. Cell Biol., 29: 291313 (1989). In such an image analysissystem, it is preferred to use a high quality CCD camera whose intensityresponse is known to be linear over a wide range of intensities.

The components of a particular quantitative image processing system(QUIPS) are described in Example 1 under the subheading FluorescenceMicroscopy and Interpretation of Results. As exemplified in Example 1, acomputer-assisted image analysis system with a filterwheel is used sothat the images from the signals and counterstaining of the DNA aresuperimposed on one image. Pseudocolors, that is, colors that are notexactly spectrally converted, can be displayed. Contrast stretching,wherein the differences between the intensity levels of the signals andbackground staining differences are enhanced by adjusting controls ofthe image analysis system. Thresholding can also be used wherein thebackground staining can be assigned a value close to zero so it wouldbarely appear in the processed image from such a system. Similarly,computer analysis permits substraction of background, smoothing offluctuations in the signals, accurate intensity and ratio calculationsand the ability to average signals on chromosomes in multiple spreads.

Absolute Copy Numbers:

Hybridization of the subject DNAs to the reference chromosomes givesinformation on relative copy numbers of sequences. Some additionalnormalization is required to obtain absolute copy number information.One convenient method to do this is to hybridize a probe, for example acosmid specific to some single locus in the normal haploid genome, tothe interphase nuclei of the subject cell or cell population(s) (orthose of an equivalent cell or representative cells therefrom,respectively). Counting the hybridization signals in a representativepopulation of such nuclei gives the absolute sequence copy number atthat location. Given that information at one locus, the intensity(ratio) information from the hybridization of the subject DNA(s) to thereference condensed chromosomes gives the absolute copy number over therest of the genome. In practice, use of more than one reference locusmay be desirable. In this case, the best fit of the intensity (ratio)data through the reference loci would give a more accurate determinationof absolute sequence copy number over the rest of the genome.

Thus, the CGH methods of this invention combined with other well-knownmethods in the art can provide information on the absolute copy numbersof substantially all RNA or DNA sequences in subject cell(s) or cellpopulations) as a function of the location of those sequences in areference genome. For example, one or more chromosome-specific repeatsequence or high complexity painting probes can be hybridizedindependently to the interphase nuclei of cells representative of thegenomic constitution of the subject cell(s) or cell population(s). Wholechromosome painting probes are now available for all the humanchromosomes [Collins et al., Genomics, 11: 997-1006 (1991)]. Specificrepeat-sequence probes are also available [Trask et al., Hum. Genet.,78: 251 (1988) and references cited therein; and commercially availablefrom Oncor (Gaithersburg, Md., USA)]. Hybridization with one or more ofsuch probes indicates the absolute copy numbers of the sequences towhich the probes bind.

For such interphase analysis, painting probes with a complexity of fromabout 35 kb to about 200 kb, are preferred; probes from about 35 kb toabout 100 kb are further preferred; and still more preferred are probeshaving a complexity of from about 35 kb to 40 kb, for example, a cosmidprobe. Exemplary of such locus-specific painting probes are any cosmid,yeast artificial chromosomes (YACs), bacterial artificial chromosomes(BACs), and/or pi phage probes as appropriate, preferably to the arms ofa selected chromosome. Such cosmid probes, for example, are commerciallyavailable from Clontech [South San Francisco, Calif. (USA)] whichsupplies cosmid libraries for all the human chromosomes. Another exampleof a cosmid probe that could be used in such methods of this inventionwould be a 3p cosmid probe called cCI3-787 obtained from YusukeNakamura, M.D., Ph.D. [Division of Biochemistry, Cancer Institute,Toshima, Tokyo, 170, Japan]. Its isolation and mapping to 3p21.2-p21.1is described in Yamakawa et al., Genomics, 1(3): 536-543 (1991). Anotherexample would be a 3q cosmid probe named J14R1A12 obtained from Wen-LinKuo [Biomedical Department, P.O. Box 5507 (L-452), Lawrence LivermoreNational Laboratory Livermore, Calif. 94550 (USA)]. For interphaseanalysis, preferred repeat sequence probes are centromeric-specificand/or peri-centromeric-specific repeat sequence probes. Such acentromeric-probe is, for example, the chromosome 17 peri-centromericrepeat probe (cosmid ck 17.10) and the alpha satellite repeat probe forthe centromeric region of chromosome 8, both of which are described inExample 1 infra. A variety of repeat sequence probes are commerciallyavailable from Oncor [Gaithersburg, Md. (USA)]. However, thelocus-specific painting probes are preferred over the repeat sequenceprobes for the methods of this invention to determine absolute copynumbers of nucleic acid sequences.

Further, when the subject nucleic acid sequences are DNA, the referencecopy numbers can be determined by Southern analysis. When the subjectnucleic acid sequences are RNA, the reference copy numbers can bedetermined by Northern analysis.

Those reference copy numbers or reference frequencies provide a standardby which substantially all the RNA or DNA sequences in the subjectcell(s) or cell population(s) can be determined. CGH methods are used todetermine the relative copy numbers of the rest of the sequences.However, absolute copy numbers require a standard against which theresults of CGH can be determined. Othervvise the CGH procedures wouldhave to be highly standardized and quantitated to see differences in theabsolute copy numbers of sequences in a genome, for example, haploidy,triploidy, octaploidy, wherein there are 1, 3 and 8 copies of each ofthe chromosomes, respectively.

PCR and Microdisgection:

The mechanics of PCR are explained in Saiki et al., Science, 230: 1350(1985) and U.S. Pat. Nos. 4,683,195, 4,683,202 (both issued Jul. 18,1987) and 4,800,159 (issued Jan. 24, 1989).] PCR offers a rapid,sensitive and versatile cell-free molecular cloning system in which onlyminute amounts of starting material are required.

A preferred PCR method to amplify the subject nucleic acids for testingby CGH is a PCR adapter-linker amplification (Saunders et al., Nuc,Acids Res., 17 9027 (1990); Johnson, Genomics, 6: 243 (1990) and PCT90/00434 (published Aug. 9, 1990).] The labeled subject nucleic acidcould be produced by such a adapter-linker PCR method from a few hundredcells; for example, wherein the subject nucleic acid is tumor DNA, thesource DNA could be a few hundred tumor cells. Such a method couldprovide a means to analyze by CGH clonal sub-populations in a tumor.

Another preferred PCR method is a method employing a mixture of primersdescribed in Meltzer et al., "Rapid Generation of Region Specific Probesby Chromosome Microdissection and their Application: A Novel Approach toIdentify Cryptic Chromosomal Rearrangements," Nature--Genetics, 1(1):24-28 (April 1992). Microdissection of sites in the reference metaphasespread that produce signals of interest in CGH, would permit PCRamplification of nucleic acid sequences bound at such sites. Theamplified nucleic acid could then be easily recovered and used to probeavailable libraries, as for example, cosmid libraries, so that theamplified sequences could be more rapidly identified.

High copy repetitive sequences can be suppressed in amplifying thesubject nucleic acid by PCR. The PCR primers used for such a procedureare complementary to the ends of the repetitive sequences. Thus, uponproper orientation, amplification of the sequences flanked by therepeats occurs. One can further suppress production of repetitivesequences in such a PCR procedure by first hybridizing complementarysequences to said repetitive sequences wherein said complementarysequences have extended non complementary flanking ends or areterminated in nucleotides which do not permit extension by thepolymerase. The non-complementary ends of the blocidng sequences preventthe blocking sequences from acting as a PCR primer during the PCRprocess. Primers directed against the Alu and Li repetitive DNA familieshave allowed the selective amplification of human sequences byinterspersed repetitive sequence PCR (IRS-PCR) [Nelson et al., PNAS, 86:6686 (1989); Ledbetter et al., Genomics, 6: 475 (1990)].

Archived Material

An important aspect of this invention is that nucleic acids fromarchived tissue specimens, for example, paraffm-embedded orformalin-fixed pathology specimens, can be tested by the methods of CGH.Said nucleic acid cannot, of course, be prepared into chromosome spreadsfor traditional cytogenetic chemical staining. Also, it is difficult forlarge enough restriction fragments to be extracted from such materialfor other conventional research tools, such as Southern analysis.However, the nucleic acid from such specimens can be extracted by knowntechniques such as those described in Greer et al., Anatomic Pathology,2(2): 117-124 (1991) and Dubeau et al., Cancer Res., 46: 2964-2969(1986), and if necessary, amplified for testing by various CGH methods.Such nucleic acid can be amplified by using a polymerase chain reaction(PCR) procedure (described above), for example, by the method describedin Greer et al., supra wherein DNA from paraffin-embedded tissues isamplified by PCR.

A particular value of testing such archived nucleic acid is that suchspecimens are usually keyed to the medical records of the patients fromwhom the specimens were taken. Therefore, valuable diagnostic/prognosticassociations can be made between the revealed cytogenetic state ofpatients' nucleic acid material and the medical histories of treatmentand outcome for those patients. For example, information gathered by CGHcan be used to predict the invasiveness of a tumor based upon itsamplification and/or deletion pattern matched to associations made withsimilar patterns of patients whose outcomes are known.

Analogously, other nucleic acid that is fixed by some method, as, forexample, archeological material preserved through natural fixationprocesses, can also be studied by CGH procedures. As indicated above,copy number differences between species provide information on thedegree of similarity and divergence of the species studied.Evolutionarily important linkages and disjunctions between and amongspecies, extant or extinct, can be made by using the methods of CGH.

Tumor cytogenetics

CGH provides the means to assess the association between geneamplification and/or deletion and the extent of tumor evolution.Correlation between amplification and/or deletion and stage or grade ofa cancer may be prognostically important because such information maycontribute to the definition of a genetically based tumor grade thatwould better predict the future course of disease with more advancedtumors having the worst prognosis. In addition, information about earlyamplification and/or deletion events may be useful in associating thoseevents as predictors of subsequent disease progression. Geneamplification and deletions as defined by CGH to, for example, normalmetaphase spreads (genomic site, intensity of the signal and/ordifferences in signal ratios, and number of different genomic sites atwhich the copy number differences occur) can be associated with otherknown parameters such as tumor grade, histology, Brd/Urd labeling index,hormonal status, nodal involvement, tumor size, survival duration andother tumor properties available from epidemiological and biostatisticalstudies. For example, tumor DNA to be tested by CGH could includeatypical hyperplasia, ductal carcinoma in situ, stage I-III cancer andmetastatic lymph nodes in order to permit the identification ofassociations between amplifications and deletions and stage.

The associations made may make possible effective therapeuticintervention. For example, consistently amplified regions may contain anoverexpressed gene, the product of which may be able to be attackedtherapeutically (for example, the growth factor receptor tyrosinekinase, p185^(HER2)).

CGH hybridizations of nucleic acids from cells of primary cancers thathave metastasized to other sites can be used to identify amplificationand/or deletion events that are associated with drug resistance. Forexample, the subject nucleic acids to be analysed could be selected sothat approximately half are from patients whose metastatic diseaseresponded to chemotherapy and half from patients whose tumors did notrespond. If gene amplification and/or deletion is a manifestation ofkaryotypic instability that allows rapid development of drug resistance,more amplification and/or deletion in primary tumors from chemoresistantpatients than in tumors in chemosensitive patients would be expected.For example, if amplification of specific genes is responsible for thedevelopment of drug resistance, regions surrounding those genes would beexpected to be amplified consistently in tumor cells from pleuraleffusions of chemoresistant patients but not in the primary tumors.Discovery of associations between gene amplification and/or deletion andthe development of drug resistance may allow the identification ofpatients that will or will not benefit from adjuvant therapy.

Once a new region of amplification or deletion has been discovered byCGH, it can be studied in more detail using chromosome-specific painting[Pinkel et al., PNAS (USA), 85: 9138-9142 (1988); EP Publication No.430,402 (Jun. 5, 1991)) with a collection of probes that span theamplified or deleted region. Probes to amplified regions will show moresignals than centromeric signals from the same chromosome, whereasprobes to nonamplified regions will show approximately the same numberof test and centromeric signals. For example, the amplified regions on17q22-23 and 20qter (discussed as newly discovered regions ofamplification in Example 1) show variability in size from tumor to tumorusing CGH (the 17q22-23 region more markedly); it can be expected thatthe region containing the important gene(s) can be narrowed by mappingthe regions of amplification in multiple tumors in more detail to findthe portion that is amplified in all cases. Probes for those studies canbe selected, for example from specific cosmid libraries produced by theNational Laboratory Gene Library Project and/or from the NationalInstitute of Health (NE) genomic research projects.

The c-erbB-2 oncogene, also referred to as HER-2 or neu, encodes for a185 Idlodalton (Kd) protein. Studies have reported c-erbB-2 geneamplification in human mammary tumor cell lines. [Kraus et al., EMBO J.6: 605-610 (1987); van de Vijver et al., Mol. Cell Biol., 7: 2019-2023(1987).] Also, c-erbB-2 gene amplification in human breast cancer hasbeen shown to be associated with disease behavior, and may be apredictor of clinical outcome. [Slamon et al., Science, 235: 177-182(1987); Berger et al., Cancer Res., 48: 1238-1243 (1988); Zhou et al.,Cancer Res., 47: 6123-6125 (1987); and Venter et al., Lancet, 11: 69-71(1987)]. C-erbB-2 has also been shown to be amplified in ovariancancers. [Alitalo and Schwab, Advances in Cancer Res., 47: 235-281(1986).]

C-myc is a proto-oncogene which is the cellular homolog of thetransforming gene of the chicken retrovirus MC29. In humans, c-myc lieson the long arm of chromosome 8, at band 124, and spans about 5 kdlobasepairs. The myc protein is a phosphoprotein present in the nucleus. Thenormal function of c-myc is unknown; however, it also certainly plays arole in cell division, and is expressed in normally growing cells aswell as in tumor cells. It is now widely believed that translocationsinvolving c-myc lead to altered transcription of the gene, contributingto malignant transformation.

Sequences from N-myc member of the mvc gene family hare been shown to beamplified as much as a thousandfold in some neuroblastomas. N-mycgamplifications are usually seen in the later stage m and IV tumors. Somesmall-cell lung carcinomas also have amplified my& genes in doubleminute chromosomes (DMs) and homogeneously staining regions (HSRs). Mychas also been shown to be amplified in colon cancer. [Alitalo andSchwab, supra.] Again such amplifications are found in late stages oftumor development, in the so-called variant cells that exhibit a moremalignant behavior. Amplifications can involve either c-myc, N-myc oranother member of the myc gene family, L-myc. [Watson et al., supra atpp. 1084-1086].

In addition, overexpression has been observed for the p-glycoproteingene family associated with multi-drug resistance and for drugmetabolizing enzymes such as P450 containing enzymes and glutathioneS-transferase. [Fairchild and Cowan, J. Radiation Oncol. Biol. Phys.,20: 361-367 (1990).]

Identification of amplified and/or deleted genes is important to themanagement of cancer, for example, breast cancer, for several reasons:

1) to improve prognostication;

2) to detect amplification and/or deletion events that are associatedwith the development of drug resistance; and

3) to improve therapy.

For example, in regard to improving prognostication, in breast cancerthe amplification of oncogenes, such as int-2, erbB-2 and myc occurfrequently and have been associated with aggressive growth and poorprognosis in some studies. [Schwab and Amier, Genes, Chromosomes &Cancer, 1: 181-193 (1990).] In regard to reason (2), gene amplificationhas clearly been shown to lead to drug resistance in vitro (for example,amplification of the dihydrofolate reductase gene confers resistance tomethotrexate), and is likely to occur in patients undergoing therapy aswell (for example, as a result of over expression of glutathioneS-transferase and p-glycoprotein). [Fairchild and Cowan, supr]. Thus,the identification of resistance-linked genes would have a major impacton therapy by allowing therapy modification as resistance-related geneamplification occurs. Therapy could be improved by targeting forspecific therapy, tumors that overexpress specific amplified genes.

Prenatal Diagnosis

Prenatal screening for disease-linked chromosome aberrations (e.g.,trisomy 21) is enhanced by the rapid detection of such aberrations bythe methods and compositions of this invention. CGH analysis isparticularly significant for prenatal diagnosis in that it yields morerapid results than are available by cell culture methods.

Removal of Repetitive Sequences and/or Disabling the HybridizationCapacity of Repetitive Sequences

The following methods can be used to remove repetitive sequences and/ordisable the hybridization capacity of such repetitive sequences. Suchmethods are representative and are expressed schematically in terms ofprocedures well known to those of ordinary skill the art, and which canbe modified and extended according to parameters and procedures wellknown to those in the art.

Bulk Procedures. In many genomes, such as the human genome, a majorportion of distributed (or shared) repetitive DNA is contained in a fewfamilies of highly repeated sequences such as Alu. These methodsprimarily exploit the fact that the hybridization rate of complementarynucleic acid strands increases as their concentration increases. Thus,if a mixture of nucleic acid fragments is denatured and incubated underconditions that permit hybridization, the sequences present at highconcentration will become double-stranded more rapidly than the others.The double-stranded nucleic acid can then be removed and the remainderused in the hybridizations. Alternatively, the partially hybridizedmixture can be used as the subject nucleic acid, the double-strandedsequences being unable to bind to the target. The following are methodsrepresentative of bulk procedures that are useful for disabling thehybridization capacity of repetitive sequences or removing thosesequences from a mixture.

Self-reassociation. Double-stranded nucleic acid in the hybridizationmixture is denatured and then incubated under hybridization conditionsfor a time sufficient for the high-copy sequences in the mixture tobecome substantially double-str-anded. The hybridization mixture is thenapplied to the reference chromosome spread. The remaining labeledsingle-stranded copies of the highly repeated sequences may bindthroughout the reference chromosome spread producing a weak, widelydistributed signal.

Use of blocking nucleic acid. Unlabeled nucleic acid sequences which arecomplementary to those sequences in the hybridization mixture whosehybridization capacity it is desired to inhibit are added to thehybridization mixture. The subject nucleic acids and blocking nucleicacid are denatured, if necessary, and incubated under appropriatehybridization conditions. The sequences to be blocked becomedouble-stranded more rapidly than the others, and therefore are unableto bind to the reference spread when the hybridization mixture isapplied to the spread. In some cases, the blocking reaction occurs soquickly that the incubation period can be very short, and adequateresults can be obtained if the hybridization mix is applied to thespread immediately after denaturation. Further, the probe and the targetcan be simultaneously denatured in some cases. A blocking method isgenerally described in the context of Southern analysis by Sealy et al.,"Removal of Repeat Sequences form Hybridization Probes", Nucleic AcidResearch, 13:1905 (1985). Examples of blocking nucleic acids includegenomic DNA, a high-copy fraction of genomic DNA and particularsequences as outlined below.

i. Genomic DNA. Genomic DNA contains all of the nucleic acid sequencesof the organism in proportion to their copy-number in the genome. Thus,adding genomic DNA to the hybridization mixture increases theconcentration of the high-copy repeat sequences more than low-copysequences, and therefore is more effective at blocking the former.

ii. Highcopy fraction of genomic DNA. Fractionating the genomic DNA toobtain only the high-copy sequences and using them for blocking can bedone, for example, with hydroxyapatite as described below.

Removal of Sequences.

Hydroxyapatite. Single- and double-stranded nucleic acids have differentbinding characteristics to hydroxyapatite. Such characteristics providea basis commonly used for fractionating nucleic acids. Hydroxyapatite iscommercially available [e.g., Bio-Rad Laboratories, Richmond, Calif.(USA)]. The fraction of genomic DNA containing sequences with aparticular degree of repetition, from the highest copy-number tosingle-copy, can be obtained by denaturing genomic DNA, allowing it toreassociate under appropriate conditions to a particular value of C_(o)t, followed by separation using hydroxyapatite. The single- anddouble-stranded nucleic acid can also be discriminated by use of S1nuclease. Such techniques and the concept of C_(o) t are explained inBritten et al., "Analysis of Repeating DNA Sequences by Reassociation",in Methods in Enzymology, 29: 363-418 (1974).

Reaction with immobilized nucleic acid. Removal of particular sequencescan also be accomplished by attaching single-stranded "absorbing"nucleic acid sequences to a solid support. Single-standed source nucleicacid is hybridized to the immobilized nucleic acid. After thehybridization, the unbound sequences are collected and used in CGH. Forexample, human genomic DNA can be used to absorb repetitive sequencesfrom the subject nucleic acids. One such method is described by Brisonet al., "General Method for Cloning Amplified DNA by DifferentialScreening with Genomic Probes," Molecular and Cellular Biology. 2:578-587 (1982). Briefly, minimaily sheared human genomic DNA is bound todiazonium cellulose or a like support. The source DNA, appropriately cutinto fragments, is hybridized against the immobilized DNA to C_(o) tvalues in the range of about 1 to 100. The preferred stringency of thehybridization conditions may vary depending on the base composition ofthe DNA.

Prehybridization. Blocking of repeat sequence binding sites in thereference genome by hybridization with unlabeled complementary sequenceswill prevent binding of labeled sequences in the subject nucleic acidsthat have the potential to bind to those sites. For example,hybridization with unlabeled genomic DNA will render the high-copyrepetitive sequences in the reference genome double-stranded. Labeledcopies of such sequences in the subject nucleic acids will not be ableto bind when they are subsequently applied.

In practice, several mechanisms can be combined to produce the desiredcontrast and sensitivity.

Labeling the Nucleic Acid Fragments of the Subject Nucleic Acids

There are many techniques available for labeling single- anddouble-stranded nucleic acid fragments of the subject nucleic acids.They include incorporation of radioactive labels, e.g. Harper et al.Chromosome, 83: 431-439 (1984); direct attachment of fluorochromes orenzymes, e.g. Smith et al., Nuc. Acids Res., 13: 2399-2412 (1985), andConnolly et al., Nuc. Acids Res., 13: 4485-4502 (1985); and variouschemical modifications of the nucleic acid fragments that render themdetectable immunochemically or by other affinity reactions, e.g. Tchenet al., "Chemically Modified Nucleic Acids as Immunodetectable Probes inHybridization Experiments," PNAS, 81: 3466-3470 (1984); Richardson etal., "Biotin and Fluorescent Labeling of RNA Using T4 RNA Ligase," Nuc.Acids Res., 11: 6167-6184 (1983); Langer et al., "Enzymatic Synthesis ofBiotin-Labeled Polynucleotides: Novel Nucleic Acid Affinity Probes,"PNAS, 78: 6633-6637 (1981); Brigati et al., "Detection of Viral Genomesin Cultured Cells and Paraffin-Embedded Tissue Sections UsingBiotin-Labeled Hybridization Probes," Virol., 126: 32-50 (1983); Brokeret al., "Electron Microscopic Visualization of TRNA Genes withFerritin-Avidin: Biotin Labels," Nuc. Acids Res., 5: 363-384 (1978);Bayer et al., "The Use of the Avidin Biotin Complex as a Tool inMolecular Biology," Methods of Biochem. Analysis. 26: 1-45 (1980);Kuhlmann, Immunoenzyme Techniques in Cytochemistry (Weinheim, Basel,1984). Langer-Safer et al., PNAS (USA), 79: 4381 (1982): Landegent etal., Exp. Cell Res., 15: 61 (1984); and Hopman et al., Exp. Cell Res.,169: 357 (1987). Thus, as indicated, a wide variety of direct and/orindirect means are available to enable visualization of the subjectnucleic sequences that have hybridized to the reference genome. Suitablevisualizing means include various ligands, radionuclides, fluorochromesand other fluorescers, chemiluminescers, enzyme substrates orco-factors, particles, dyes and the like. Some preferred exemplarylabeling means include those wherein the probe -fragments arebiotinylated, modified with N-acetoxy-N-2-acetylaminofluorene, modifiedwith fluorescein isothiocyanate or other fluorochromes, modified withmercury/TNP ligand, sulfonated, digoxigeninated or contain T--T dimers.

A preferred method of labeling is tailing by terminal transferaselabeling. Another preferred method is random priming with mixed sequenceprimers followed by polymerase extension. This has the additionalfeature of amplifying the amount of subject DNA, if several cycles areused, which is useful when only a small amount of DNA was originallyobtained from the subject cell or cell population.

The key feature of labeling is that the subject nucleic acid fragmentsbound to the reference spread be detectable. In some cases, an intrinsicfeature of the subject nucleic acid, rather than an added feature, canbe exploited for this purpose. For example, antibodies that specificallyrecognize RNA/DNA duplexes have been demonstrated to have the ability torecognize probes made from RNA that are bound to DNA targets (Rudkin andStollar, Nature, 265;472-473 (1977)]. The RNA used is unmodified.Nucleic acid fragments can be extended by adding "tails" of modifiednucleotides or particular normal nucleotides. When a normal nucleotidetail is used, a second hybridization with nucleic acid complementary tothe tail and containing fluorochromes, enzymes, radioactivity, modifiedbases, among other labeling means, allows detection of the bound nucleicacid fragments. Such a system is commercially available from EnzoBiochem [Biobridge Labeling System; Enzo Biocliem Inc., New York, N.Y.(USA)].

Another example of a means to visualize the bound nucleic acid fragmentswherein the nucleic acid sequences do not directly carry some modifiedconstituent is the use of antibodies to thymidine dimers. Nakane et al.,ACTA Histochem. Cytochem., 20 (2):229 (1987), illustrate such a methodwherein thymine-thymine dimerized DNA (T--T DNA) was used as a markerfor in situ hybridization. The hybridized T--T DNA was detectedimmunohistochemically using rabbit anti-T--T DNA antibody.

All of the labeling techniques disclosed in the above references may bepreferred under particular circumstances. Further, any labelingtechniques known to those in the art would be useful to label thesubject nucleic acids in of this invention. Several factors govern thechoice of labeling means, including the effect of the label on the rateof hybridization and binding of the nucleic acid fragments to thechromosomal DNA, the accessibility of the bound nucleic acid fragmentsto labeling moieties applied after initial hybridization, the mutualcompatibility of the labeling moieties, the nature and intensity of thesignal generated by the label, the expense and ease in which the labelis applied, and the like.

Several different subject nucleic acids, each labeled by a differentmethod, can be used simultaneously. The binding of different nucleicacids can thereby be distinguished, for example, by different colors.

In Situ Hybridization.

Application of the subject nucleic acids to the reference chromosomespreads is accomplished by standard in s hybridization techniques.Several excellent guides to the technique are available, e.g., Gall andPardue, "Nucleic Acid Hybridization in Cytological Preparations,"Methods in Enzymology, 21: 470-480 (1981); Henderson, "CytologicalHybridization to Mammalian Chromosomes," International Review ofCytology. 76: 146 (1982); and Angerer et al., "in situ Hybridization toCellular RNAS," in Genetic Engineering; Principles and Methods, Setlowand Hollaender, Eds., Vol. 7, pgs. 43-65 (Plenum Press, New York, 1985).

Generally in situ hybridization comprises the following major steps: (1)fixation of tissue or biological structure to be examined, (2)prehybridization treatment of the biological structure to increaseaccessibility of target DNA, and to reduce nonspecific binding, (3)hybridization of the mixture of nucleic acids to the nucleic acid in thebiological structure or tissue; (4) posthybridization washes to removenucleic acid fragments not bound in the hybridization and (5) detectionof the hybridized nucleic acid fragments. The reagents used in each ofthese steps and their conditions of use vary depending on the particularsituation.

Under the conditions of hybridization wherein human genomic DNA is usedas an agent to block the hybridization capacity of the repetitivesequences, the preferred size range of the nucleic acid fragments isfrom about 200 bases to about 1000 bases, more preferably about 400 to800 bases for double-stranded, nick-translated nucleic acids and about200 to 600 bases for single-stranded or PCR adapter-linker amplifiednucleic acids.

Example 1 provides details of a preferred hybridization protocol.Basically the same hybridization protocols as used forchromosome-specific painting as described in Pinkel et al., PNAS (USA),85: 9138-9142 (1988) and in EP Pub. No. 430,402 (published Jun. 5, 1991)are adapted for use in CGH.

The following representative examples of performing CGH methods of thisinvention are for purposes of illustration only and are not meant tolimit the invention in any way.

EXAMPLE 1 DNA from Breast Cancer Lines Hybridized to Normal MetaphaseSpreads

In this Example, methods of this invention to analyze genomes byComparative Genomic Hybridization (CGH) are exemplified byhybridizations of breast cancer cell lines to normal metaphase spreads.The target metaphase spreads were pre-hybridized with unlabeled humanplacental DNA to block the high copy repeat ;equences. In thisrepresentative example, the hybridization mixture containing theextracted labeled DNA from the cell lines contained unlabeled,repeat-enriched Cot-1 blocking DNA [obtained from Bethesda ResearchLaboratories (BRL), Gaithersburg, Md. (USA)].

The experiments outlined below include in the hybridization mixture forthe subject genomes, that is, the breast cancer cell line DNAS,chromosome-specific repeat sequence probes and chromosome-specificpainting probes. Those probes labeled with biotin were included as anadjunct for identifying chromosomes in the metaphase preparations. Theexperiments were first performed without those chromosome-specificprobes. Then each chromosome of interest was measured to determine itslength which was considered along with other factors to determine itsprobable identity. The chromosome-specific probes were then used in thehybridization mixture to confirm the identity of the chromosome ofinterest. However, such probes are not necessary as the chromosomescould have been identified by the DAPI banding of the counterstain or byother chemical staining, such as staining with quinacrine, by a skilledcytogeneticist.

Cell Lines and Isolation of DNA:

Six established breast cancer cell lines: BT474, SK-BR-3, MCF-7,MDA-MB-361, MDA-MB468 and T47D were obtained from the American TypeCulture Collection (Rockville, Md. (USA)]. The breast cancer cell line600MPE cell line was kindly provided by Dr. Helene S. Smith [GeraldineBrush Cancer Research Center, San Francisco, Calif. (USA)]. Cell lineswere grown until they became confluent. Cells were then trypsinized,pelleted by centrifugation at 1500 RPM for 5 minutes and washed twice inphosphate buffered saline. The DNA was then isolated as described bySambrook et al., Molecular Cloning; A Laboratory Manual, Vol. 2:9.16-9.19 [Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(USA) 1989].

Details concerning the established human breast cancer cell lines usedherein are as follows:

    ______________________________________                                        BT-474 originated from a human primary cancer; obtained from the                 ATCC, catalog # HTB 20;                                                      SK-BR-3 Originated from a human metastatic breast adenocarcinoma                     derived from a pleural effusion; obtained from the ATCC                 catalog # HTB 30;                                                            MDA- originated as a metastadc tumor to the brain; obtained from                    MB-361 the ATCC, catalog # HTB 27;                                      MCF-7 originated from a human metastatic pleural effusion; obtained                  from the ATCC, catalog HTB 22;                                         T-47D originated as a human metastatic pleural effusion; obtained                    from the ATCC catalog HTB 133;                                         60OMPE originated as a human metastatic pleural effusion; kindly                     provided by Dr. Helene S. Smith [Geraldine Brush Cancer                 Research Center, San Francisco, CA (USA)]; and                               MDA- originated as a-metastatic pleural effusion; obtained from the                 MB-468 ATCC, catalog # HTB 132.                                       ______________________________________                                    

Preparation of Normal Lymphocyte Metaphases:

Normal peripheral blood lymphocytes were stimulated by PHA, synchronizedby methotrexate treatment and blocked in metaphase using 0.05 ug/mlcolcemid. Cells were then centrifuged, washed and incubated in 75 mM KC1at 370° C. for 15 minutes. Cells were then fixed in methanol:acetic acid(3:1) and dropped onto slides. The slides were stored under nitrogen at-200° C.

DNA Labeling:

Cell line DNAs were labeled with digoxigenin-11dUTP using nicktranslation [Rigby et al., J. Mol. Biol., 11: 237 (1977); Sambrook etal., supra]. The optimal size of the probe fragments after nicktranslation and before denaturing was 400-800 bps. As indicated above,chromosome-specific probes were used in dual-color hybridizations toverify the identification of chromosomes of interest in the metaphasespreads. Representative examples of such chromosome-specific referenceprobes labeled with biotin-14dATP include the following:

1) a chromosome-specific painting probe for chromosome 20 prepared bythe PCR adapter-linker method as described in PCTIUS90/00434 publishedAug. 9, 1990;

2) a chromosome 17 peri-centromeric repeat probe (cosmid ck 17.10)isolated by Anne Kallioniemi from a chromosome 17 cosmid library fromLos Alamos National Laboratory (Albuquerque, N.Mex. (USA)]; anequivalent chromosome-specific repeat sequence probe for chromosome 17is commercially available from Oncor [Gaithersburg, Md. (USA)]; and

3) an alpha satellite repeat probe specific for the centromeric regionof chromosome 8 [kindly provided by Dr. Heinz-Ulrich G. Weier;University of California Medical Center, Lab for Cell Analysis, SanFrancisco, Calif. (USA)]; that probe was generated by Dr. Weier usingPCR with primers WA1 and WA2 as described in Weier et al., Hum. Genet.,87: 489-494 (1991).

Ones skilled in the art recognize that there are many other equivalentprobes available that could be used for the confirmation purposesdescribed. For example, whole chromosome painting probes are nowavailable for all the human chromosomes [Collins et al., Genomics, 11:997-1006 (1991)]. Also available are repeat sequence probes thathybridize intensely and specifically to selected chromosomes (Trask etal., Hum. Genet., 78: 251 (1988) and references cited therein].

Pretreatment and Prehybridization of Slides:

Lymphocyte metaphase preparations were first denatured in 70%formamide/2×SSC (1×SSC is 0.15 M NaCl, 0.015 M NaCitrate), pH 7, at 70°C. for 2 minutes and dehydrated in a sequence of 70%, 85% and 100%ethanol. The slides were then air dried and treated with 10 ug/50 mlProteinase K [Boehringer Mannheim GmbH, Indianapolis Ind. (USA)] for 7.5minutes at 37° C. in a buffer containing 20 mM Tris and 2 mM CaCI₂ (pH7.5). Ethanol dehydration was then done as described above, and theslides were prehybridized with ten ul of a hybridization mixture,consisting of 20 ug unlabeled human placental DNA [obtained from sigma,St. Louis, Mo. (USA); size of the fragments is 200-700 bps] in 50%formamide, 10% dextran sulphate and 2×SSC (pH 7) for 60 minutes at 37°C. Before the prehybridization mixture was applied to the slides, it wasdenatured in a 70° C. water bath for 5 minutes. After prehybridization,the slides were washed once in 2×SSC and dehydrated with ethanol asdescribed above.

Hybridization:

Five ug of unlabeled, repeat-enriched Cot-1 blocking DNA [BRL,Gaithersburg, Md. (USA)] and 60 ng of digoxigenin labeled cell line DNAand 20-60 ng of biotinlabeled reference probes (for verification ofchromosome identification) were mixed together and 1/10 vol of 3MNaacetate was added. DNA was precipitated by adding 2 volumes of 100%ethanol followed by centrifugation in a microcentrifuge for 30 minutesat 15,000 RPM. Ethanol was removed and the tubes were allowed to dryuntil all visible ethanol had evaporated. Ten ul of hybridization bufferconsisting of 50% formamide, 10% dextran sulphate and 2×SSC (pH 7) wasthen added, followed by careful mixing. DNAs in the hybridization bufferwere then denatured for 5 minutes at 70° C. followed by a 60 minuterenaturation at 37° C. The hybridization mixture was then added to theprehybridized lymphocyte metaphase slides. Hybridization was carried outunder a coverslip in a moist chamber for 3-4 days at 37° C.

Immunofluorescent Probe Detection:

The slides were washed three times in 50% formamide/2×SSC, pH 7, twicein 2×SSC and once in 0.1×SSC for 10 minutes each at 450° C. Afterwashing, the slides were immunocytochemically stained at roomtemperature in three steps (30-45 minutes each). Before the firstimmunocytochemical staining, the slides were preblocked in 1% BSA/4×SSCfor 5 minutes. The first staining step consisted of 2 ug/ml TexasRed-Avidin [Vector Laboratories, Inc., Burlingame, Calif. (USA)] in 1%BSA/4×SSC. The slides were then washed in 4×SSC, 4×SSC/0.1% TritonX-100, 4×SSC, and PN (a mixture of 0.1 M NaH₂ PO₄ and 0.1 M Na₂ HPO₄, pH8, and 0.1% Nonidet P-40) for 10 minutes each and preblocked with PNM(5% Carnation dry milk, 0.02% Na-azide in PN. buffer) for 5 minutes. Thesecond antibody incubation consisted of 2 ug/ml FITC-conjugated sheepanti-digoxigenin [Boehringer Mannheim GMBH, Indianapolis, Ind. (USA)]and 5 ug/mi anti-avidin [Vector Laboratories, Burlingame, Calif. (USA)]in PNM followed by three PN washes, 10 minutes each. After the PNMblock, the third immunochemical staining was done using rabbitanti-sheep FITC antibody (1:50 dilution) (Vector Laboratories) and 2ug/ml Texas Red-Avidin in PNM. After three PN washes, nuclei werecounterstained with 0.8 uM 4,5-diamino-2-phenylindole (DAPI) in anantifade solution.

Fluorescence Microscopy and Interpretation of Results:

A Nikon fluorescence microscope [Nikon Inc., Garden City, N.Y. (USA)]equipped with a double band pass filter (Chroma Technology, Brattleboro,Vt. (USA)] and a 100× objective was used for simultaneous visualizationof the FITC and Texas Red signals. Hybridization of the breast cancercell line DNAs was seen as a more or less uniform faint green backgroundstaining of all metaphase chromosomes with the exception of theY-chromosome. As the breast cancer cell lines are of course of femaleorigin, they did not contain Y chromosomal DNA. The absence of saidgreen staining of the Y chromosome of the metaphase spread (seen in FIG.8) is exemplary of the manner in which a cytogenetically significantdeletion would be visualized. Using a fluorescence microscope, amplifiedsequences can be seen as bright green dots or bands along the chromosomearms.

To facilitate the display of the results and to improve the sensitivityof detecting small differences in fluorescence intensity, a digitalimage analysis system (QUIPS) was used. QUIPS (an acronym forquantitative image processing system) is an automated image analysissystem based on a standard Nikon Microphot SA [Nikon Inc., Garden City,N.Y. (USA)] fluorescence microscope equipped with an automated stage,focus control and filterwheel [Lud1 Electronic Products Ltd., Hawthorne,N.Y. (USA)]. The filterwheel is mounted in the fluorescence excitationpath of the microscope for selection of the excitation wavelength.Special filters [Chroma Technology, Brattleboro, Vt. (USA)] in thedichroic block allow excitation of multiple dyes without imageregistration shift. The microscope has two camera ports, one of whichhas an intensified CCD camera [Quantex Corp., Sunnyvale, Calif. (USA)]for sensitive high-speed video image display which is used for findinginteresting areas on a slide as well as for focusing. The other cameraport has a cooled CCD camera [model 200 by Photometrics Ltd., Tucson,Ariz. (USA)] which is used for the actual image acquisition at highresolution and sensitivity.

The cooled CCD camera is interfaced to a SUN 4/330 workstation [SUNMicrosystems Inc., Mountain View, Calif. (USA)] through a VME bus. Theentire acquisition of multicolor images is controlled using an imageprocessing software package SCIL-Image [Delft Centre for ImageProcessing, Delft, Netherlands]. Other options for controlling thecameras, stage, focus and filterwheel as well as special programs forthe acquisition and display of multicolor images were developed at theDivision of Molecular Cytometry [University of California, MedicalCenter; San Francisco, Calif. (USA)] based on the SCIL-Image package.

To display the results of the comparative hybridization, two or threeconsecutive images were acquired (DAPI, FITC and Texas Red) andsuperimposed. The FITC image was displayed after using the thresholdingand contrast enhancement options of the SCIL-Image software. Exercisingsuch options reduces the overall chromosomal fluorescence to makeamplified sequences more readily visible. For example, usingthresholding and contrast stretching, it was possible to enhance thecontrast and quantification between the faint green background stainingand staining originating from the amplified sequences in the cell lines.Alternatively, to facilitate the detection of deletions, it is possibleto increase the overall chromosomal fluorescence and make areas ofreduced fluorescence appear darker. The red color was used for referenceprobes to help in the identification of chromosomes.

After identification of the chromosomes based on the use of referenceprobes in a dual-color hybridization, a site of amplification waslocalized by fractional length measurements along the chromosome arm(fractional length distance of the hybridization signal from thep-telomere divided by the total length of the chromosome). The bandlocation of the signal was then approximated from the fractional lengthestimate based on the ISCN 1985 ideograms [Harnden and Klinger, AnInternational Svstem for Cytogenetic Nomenclaturg, Karger Ag, Basel,Switzerland (1985)].

RESULTS

The results from the hybridizations are compiled in Table 2 along withother information known about the cell lines. Amplification at 17q12(erbB-2 locus) and approximately 8q24 (MYC locus) was seen in linesshowing amplification of erbB-2 and MYC whenever the level ofamplification was greater than about five- to ten-fold using this CRCCmethod. In addition, amplification of several megabase wide regions wasseen in three cell lines at 17q2223 and in three lines at 20qter; thoseamplifications were previously unknown sites of amplification and werenot expected from other studies. All lines showing amplification showedamplification at more than one site. Evidence for co-amplification maybe clinically important since co-amplification has been observedpreviously [van de Vijver et al., Mol. Cell Biol. Z: 2019-2023 (198i);SaintRuf et al., Oncogene, 6: 403-406 (1991)], and is sometimesassociated with poor prognosis [Borg et al., Br. J. Cancer. 13: 136-142(1991)]. Amplification at 17q22-23 has also been seen using probe DNAfrom primary tumors.

                  TABLE 2                                                         ______________________________________                                        Results of Testing Breast Cancer Cell Lines for Amplification                                             Hor-          Amplifica-                              Growth mone Known tion                                                        rate; receptor amplifica- detected                                          Cell Line Origin -TD E/P tion (level) by CGH                                ______________________________________                                        BT474   Primary   48-     +/-   erbB-2  17q12                                    cancer 96 hr  (13×) (erbB-2),                                               17q22-23,                                                                     2Oqter                                                                   SK-BR-3 Pl. Effusion ? ? erbB-2 (9×) 17q12                                   (erbB-2),                                                                     8q21,                                                                        MYC (10×) 8q23-24.1                                                      (MYC),                                                                        20qter                                                                   MDA-MB- Brain met. <96 hr -/+ erbB-2 (4×) 17q-22-23                     361                                                                           MCF-7 Pl. Effusion <48 hr +/+ erbB-2 17q22-23,                                    (none) 20qter                                                             T-47D Pl. Effusion ? +/+ erbB-2 None                                              (none)                                                                    600 MPE Pl. Effusion ? ? erbB-2 None                                              (none)                                                                    MDA-MB- Pl. Effusion ? ? erbB-2 None                                          468    (none)                                                               ______________________________________                                    

EXAMPLE 2

Hybridizations with two different labeled subject DNAs as schematicallyoutlined in FIGS. 6 and 7 were performed. One of the labeled subjectDNAs hybridized was a cell line DNA as described in Example 1 andsimilarly labeled. The other labeled subject DNA was human genomic DNAlabeled with biotin-14dATP.

The protocols were essentially the same as in Example 1 except that nochromosome-specific reference probes were used, and the same amount ofthe labeled human DNA as the labeled cell line DNA, that is, 60 ng, washybridized. Of course, reference probes could be added to thehybridization mixture, but they need to be differently labeled to bedistinguishable.

The results showed the normal DNA with a red signal and the cell lineDNA with a green signal. The green to red ratios were determined alongeach chromosome. Amplification was indicated by an area where the signalwas predominantly green whereas deletions were indicated by more redsignals than in other areas of the chromosomes.

Exemplary, CGH results using breast cancer cell line 600MPE DNA andnormal human DNA were as follows. As indicated above, the hybridizationwas performed using 5 ug Cot-1 DNA, 60 ng of digoxigenin labeled 600MPEcell line DNA, and 60 ng of biotinylated normal human genomic DNA. The600MPE DNA was detected with FITC (green) and the genomic DNA with TexasRed-Avidin (red).

The 600MPE breast cancer cell line, the karyotype for which waspublished by Smith et al., JNCI, 78: 611-615 (1987), contains one normalchromosome 1 and three marker chromosomes with chromosome 1 material inthem: t(1q: 13q), 1p(p22) and inv(1)(p36q21). Thus, the cell line isdisomic for the p-telomere-p22, trisomic for p22-centromere andtetrasomic for the q-arm of chromosome 1. An idiogram of chromosome 1showing those different areas is illustrated in FIG. 9.

The comparative genomic hybridizations of this example apparentlyidentified three different regions on chromosome 1 that could beseparated according to the intensities of green and red colors. Theq-arm of chromosome 1 had the highest intensity of green color (tumorDNA). The region from band p22 to the centromere was the secondbrightest in green, and the area from the p-telomere to band p22 had thehighest intensity of red color (normal DNA). Those hybridization resultswere consistent with the traditional cytogenetic analyses of that cellline stated immediately above.

However, further studies with CGH, as presented in Example 3, indicatedthat CGH analysis of Example 2, as well as the published karyotype, werepartially in error. The CGH analysis of Example 3 motivated additionalconfirmatory experiments, as described therein, leading to correction ofthe original CGH results and the published karyotpe.

EXAMPLE 3 Copy Number Karvotypes of Tumor DNA

In the representative experiments of CGH in this example, biotinylatedtotal tumor DNA (cell line and primary tumor DNA) anddigoxigenin-labeled normal human genomic DNA are simultaneouslyhybridized to normal human metaphase spreads in the presence ofunlabeled blocking DNA containing high-copy repetitive sequences,specifically unlabeled Cot-1 blocking DNA [BRL, Gaithersburg, Md.(USA)]. The following paragraphs detail the procedures used for therepresentative CGH experiments of this example.

DNA Labeling:

DNAs used in this example were labeled essentially as shown above inExample 1. DNAs were labeled with biotin-14-dATP or digoxigenin-11-dUTPby nick translation [Rigby et at., supra: Sambrook et al., supra]. Theoptimal size for double stranded probe fragments after labeling was600-1000 bp.

Pretreatment of Metaphase Spreading:

Lymphocyte metaphase preparations were denatured, dehydrated and airdried, treated with Proteinase K and dehydrated again as described inExample 1.

Comparative Genomic Hybridization:

Sixty ng of biotinylated test DNA, 60 ng of digoxigenin-labeled normalDNA and 5 gg of unlabeled Cot-1 DNA (BRL) were ethanol precipitated anddissolved in 10 μl of 50% formamide, 10% dextran sulfate, 2xSSC, pH 7.The probe mixture was denatured at 70° C. for 5 minutes, allowed toreanneal at 37° C. for 60 minutes and hybridized to normal malemetaphase chromosomes for 3-4 days at 37° C.

Immunofluorescent Probe Detection:

The slides were washed as described above in Example 1, andimmunocytochemically stained at room temperature in three thirty-minutesteps: (I) 5 μl/ml FITCAvidin [Vector Laboratories, Inc., Burlingame,Calif. (USA)] and 2 μl/ml anti-digoxigenin-Rhodamine (BoehringerMannheim GMBH); (II) 5 μg/ml anti-avidin (Vector Laboratories); and (IIM5 μg/ml FITC-avidin. Nuclei were counterstained with 0.8 μM4,5-diamino-2-phenylindole (DAPI) in antifade solution. A Zeissfluorescence microscope equipped with a double band pass filter [ChromaTechnology, Brattleboro, Vt. (USA)] was used for simultaneousvisualization of FITC and rhodamine signals.

Digital Image Analysis System and Fluorescence Ratio Profiles

The QUIPS system essentially as described above in Example 1 was used toanalyze quantitatively the fluorescence signals. Fluorescence ratioprofiles along the chromosomes were extracted using WOOLZ softwarepackage [developed at MRC, Edinburgh, Scotland] as follows: the DAPIimage is used to set the morphological boundary of each chromosome bythresholding. The chromosome outline is smoothed by a n number ofopening and closing operations, a modified Hilditch skeleton iscalculated and taken to represent the medial axis of the chromosome. TheDAPI image is expanded outwards in all directions until the intensityfield levels off (when background is reached) or begins to rise (due toan adjacent chromosome). The intensity profile of each image along themedial axis and within the expanded DAPI image is then calculated bysumming the green and red fluorescence pixel values along the sequenceof lines perpendicular to and spaced at unit distance along the medialaxis. Modal green and red intensity values corresponding to the expandedDAPI image are taken to represent the background fluorescence and usedas the intensity origin.

Cell Lines:

5637--Originated from a human primary bladder carcinoma; obtained fromATCC, catalog # HTB 9

SK-BR-3--Originated from a human metastatic breast adenocarcinoma,derived from a pleural effusion; obtained from the ATCC, catalog # HTB30

Colo 205--originated from a human colon adenocarcinoma; obtained fromthe ATCC, catalog # CCL 222

NCI-H508--originated from a human cecum adenocarcinoma; obtained fromthe ATCC, catalog # CCL 253

SW480--Originated from a human colon adenocarcinoma; obtained from theATCC, catalog # CCL 228

SW620--Originated from a human lymph node metatasis of a colonadenocarcinoma; obtained from the ATCC, catalog # CCL 227

WiDr--originated from a human colon adenocarcinoma; obtained from theATCC, catalog # CCL 218

SK-N-MC--Originated from a human neuroblastoma (metastasis tosupra-orbital area); obtained from the ATCC, catalog # HTB, 10

CaLu3--Originated from a human lung adenocarcinoma, derived from apleural effusion; obtained from the ATCC, catalog # HTB 55

CaLu6--originated from a human anaplastic carcinoma, probably lung;obtained from the ATCC, catalog # HTB 56

NCI-H69--Originated from a human small cell lung carcinoma; obtainedfrom the ATCC, catalog # HTB 119

COLO 320HSR originated from a human colon adenocarcinoma; obtained fromthe ATCC, catalog # 220.1

600PE--Originated from a human breast carcinoma; obtained from Dr.Helene Smith and Dr. Ling Chen [Geraldine Brush Cancer Research Center,San Francisco, Calif. (USA)]. This is the same as the 600 MPE cell linedescribed in Examples 1 and 2.

BT-20--originated from a human breast carcinoma; obtained from ATCC,catalog # HTB 19

The following are five fibroblast cell lines with total chromosomalnumber and X chromosomal number in parentheses, which were obtained fromthe NIGMS repository [Camden, N.J. (USA)]:

GM01723 (45,XO)

GM08399 (46,XX)

GM04626 (47,XXX)

GM01415E (48,XXX)

GM05009B (49,XXXXX).

RESULTS AND DISCUSSION

Demonstrated herein is CGH's capability of detecting and mappingrelative DNA sequence copy number between genomes. A comparison of DNAsfrom malignant and normal cells permits the generation of a "copy numberkaryotype" for a tumor, thereby identifying regions of gain or loss ofDNA.

Demonstrated is the use of dual color fluorescence in situ hybridizationof differently labeled DNAs from a subject tumor genome and a normalhuman genome to a normal human metaphase spread to map DNA sequence copynumber throughout the tumor genome being tested. Regions of gain or lossof DNA sequences, such as deletions, duplications or amplifications, areseen as changes in the ratio of the intensities of the two fluorochromes(used in this representative example) along the target chromosomes.Analysis of tumor cell lines and primary bladder tumors identified 16different regions of amplification, many in loci not previously known tobe amplified. Those results are shown in Table 3 below.

The tumor DNA is detected with the green fluorescing FIlC-avidin, andthe normal DNA with the red fluorescing rhodamine anti-digoxigenin. Therelative amounts of tumor and normal DNA bound at a given chromosomallocus are dependent on the relative abundance of those sequences in thetwo DNA samples, and can be quantitated by measurement of the ratio ofgreen to red fluorescence. The normal DNA in this example serves as acontrol for local variations in the ability to hybridize to targetchromosomes. Thus, gene amplification or chromosomal duplication in thetumor DNA produces an elevated green to red ratio, and deletions orchromosomal loss cause a reduced ratio. The Cot-1 DNA included in thehybridization inhibits binding of the labeled DNAs to the centromericand heterochromatic regions so those regions are excluded from theanalysis.

The fluorescence signals were quantitatively analyzed by means of adigital image analysis system as described above. A software programintegrated the green and red fluorescence intensities in stripsorthogonal to the chromosomal axis, subtracted local background, andcalculated intensity profiles for both colors and the green to red ratioalong the chromosomes.

The ability of CGH to quantitate changes in sequence copy number thataffect an entire chromosome was tested with the above-listed fivefibroblast cell lines having 1 to 5 copies of the X chromosome and twocopies of each autosome. Hybridization of DNA from the 45,XO cell line(in green) together with normal female DNA (in red) resulted in auniform green-red staining of the autosomes whereas the X chromosomeappeared more red (FIG. 10A). Hybridizations with DNA from cell linescarrying 2, 3, 4 or 5 copies of the X chromosome resulted in anincreasingly strong green fluorescence from the X chromosome in relationto the autosomes. The average green-to-red fluorescence ratio of the Xchromosome (FIG. 10B), when normalized to the average ratio for theautosomes within the same metaphase spread, increased linearly with theincreasing number of X chromosomes [correlation coefficient (r)=0.978].Thus, CGH can quantitatively distinguish a change of plus or minus onecopy of a chromosome at least up to 4 copies.

Experiments showed that CGH could generate a complete copy numberkaryotype for a near-diploid breast cancer cell line, 600PE. Accordingto the published karyotype for 60OPE (Smith et al., JNCI, 78: 611(1987)], 600PE is neardiploid with five marker chromosomes having fourcopies of the q-arm of chromosome 1, monosomy 16, and deletions of 9p,11q and 17p. CGH using biotinylatel 600PE DNA (in green) and normaldigoxigenin-labeled DNA (in red) revealed the following relative copynumber changes: gain of 11q and loss of 9p, 16q, 17p and distal 11q. Thegreen to red ratio profiles for those aberrant chromosomes are shown inFIG. 11. Only the q-arm of chromosome 16 showed decreased relative copynumber suggesting that 16p was not deleted. That observation wassubsequently confirmed by fluorescence in situ hybridization (FISH) to600PE interphase cells using cosmid probes for the p- and q-arms ofchromosome 16 [16p and 16q cosmid probes provided by Los Alamos NationalLaboratory, Los Alamos, N.Mex. (USA)]; two signals per nucleus for the16p cosmid probe and one for the 16q cosmid probe permitted calibrationof a green-to-red ratio of 1.0 as indicating two copies of a sequence.

Thus, if the absolute copy number of any point in the tumor genome isknown, relative copy numbers can be converted to actual copy numbers atall loci. The CGH results differed from the originally publishedkaryotype in the region of 16p and proximal 1p. That discrepancy wasresolved by locus-specific chromosome-specific painting (FISH) thatindicated that the components of one of the marker chromosomes had beenmisinterpreted by conventional cytogenetic analysis.

CGH with DNAs from two fibroblast cell lines [GM05877 and GM01142A fromthe NIGMS repository] detected small interstitial deletions around theRB1 locus in 13q-del(13) (pter>q14.1::q21.2>qter) and del(l3)(pter>q14.1::q22.1>qter). On the basis of the CGH analysis andmeasurement of the deletion size as a fraction of the length ofchromosome 13 (total length 111 Mb), those deletions were estimated tospan about 10 and 20 megabases (Mb), respectively. Thus it is possiblethat CGH can be used to screen DNA samples from solid tumors in order toidentify large physical deletions that may uncover recessive mutanttumor suppressor genes.

CGH was evaluated for its ability to detect increased gene copy numberwith cell lines that contained previously reported amplification ofoncogenes. FIG. 12A shows CGH with DNA from a colon cancer cell lineCOLO 320HSR, known to contain more than a 50-fold amplification of a 300kb region around the myc oncogene [Kinzku et al., PNAS (USA), 83; 1031(1986)]. The expected high green to red ratio at 8q24 corresponding tothe location of myc is clear. The height of the peak does notquantitatively reflect the level of amplification because thefluorescent signal spread over a region of the chromosome that is largerthan the length of the amplicon. That is apparently a result of thecomplex organization of the target DNA in the denatured chromosomes.

The eight-fold amplification of the erbB2 oncogene in the SK-BR-3 breastcancer cell line also was detectable with CGH as a hybridization signalat 17q12 (Table 3). High level amplifications such as those also couldbe detected in single color-hybridizations with the use of only labeledtumor DNA.

Cytogenetic and molecular studies of primary tumors and cell lines oftenreveal homogeneously staining regions and double minute chromosomes thatdo not involve known oncogenes [Saint-Ruf et al., Genes Chrom. Cancer.,2: 18 (1990); Bruderlein et al., Genes Chrom. Cancer, 2: 63 (1990)]. CGHallows straightforward detection and mapping of such sequences. Table 3contains a summary of the analysis with CGH of 11 cancer cell lines.Data in Table 3 is based on the visual inspection of a large number ofmetaphase spreads and on detailed digital image analysis of four to sixmetaphases for each sample.

                  TABLE 3                                                         ______________________________________                                        Mapping of amplified sequences in established cancer                            cell lines and primary tumors by CGH                                                                            Cytogenetic                                    evidence of                                                                Specimen Origin Amplif. by CGH* gene ampli +                                ______________________________________                                        Cell lines:                                                                     5637 Bladder 3p25, 6p22 DM                                                    SK-BR-3 Breast 8q24 (mvc), 8q21,                                                17q12 (erbB2),                                                                20q13                                                                       Colo 205 Colorectal 6p21, 6q24                                                NCI-H508 Colorectal 14q12-13 DM                                               SW480 Colorectal 8q24 (myc) DM                                                SW620 Colorectal 16q21-23 HSR                                                 WiDr Colorectal 8q23-24 (myc)                                                 SK-N-MC Neuroblastoma 8q24 (myc) DM                                           CaLu3 Small cell lung 8p12-21 8qtel HSR                                         mq12 (erbB2)                                                                CaLu6 Small cell lung 13q32-34                                                NCI-H69 Small cell lung 2p24 (N-myc),                                           2p21, 2q21                                                                  Primary                                                                       tumors:                                                                       UR140 Bladder carcinoma 16q21-22                                              UR145 Bladder carcinoma 6p22                                                ______________________________________                                         *The oncogene most likely involved in this amplification is shown in          parentheses.                                                                  + Cytogenetic information based on the ATCC Catalogue of Cell Lines &         Hybridomas (1992).                                                            DM = double minute chromosornes, HSR = homogeneously staining regions.   

Sixteen amplified loci were mapped, many at regions of the genome whereamplification had not previously been suspected. Thus, a large varietyof genes may be amplified during cancer initiation and progression. Infive of the 11 cell lines, more than one locus was amplified. Two orthree separate loci on the same chromosome were amplified in four celllines, which suggests a spatial clustering of chromosomal locations thatundergo DNA amplification (Table 3 and FIG. 12A).

CGH was also applied to identify and map amplified DNA sequences inuncultured primary bladder tumors. Of the seven tumors tested, twoshowed evidence of DNA amplification but the loci were not the same(Table 3). Thus, a number of previously unsuspected genomic regions thatmight contain genes important for cancer progression have beenidentified by CGH. Further studies will elucidate which of those locicontain novel oncogenes and which represent coincidental, random DNAamplification characteristic of genomic instability.

The detection and mapping of unknown amplified sequences that typicallyspan several hundred kilobases (kb) to a few Mb demonstrated theusefulness of CGH for rapid identification of regions of the genome thatmay contain oncogenes. Analogously, detection of deletions mayfacilitate identification of regions that contain tumor suppressor.

Further studies are necessary to establish to what extent allelic lossesin tumors are caused by physical deletions. In clinical specimens, thedetection of small copy number differences is more difficult than withcell lines because of the admixture of DNA from contaminating normalcells and because of intratumor heterogeneity. As indicated above, usingPCR to prepare tumor DNA from a small number of tumor cells (as a tumorclonal sub-population) may assist in resolving that problem. Like RFLP,CGH emphasizes the detection of aberrations that are homogeneous in acell population and averages those that are heterogeneous.

At the current stage of development of CGH, sensitivity is primarilylimited by the granularity of the hybridization signals in the metaphasechromosomes. Further improvements in sensitivity will be achieved byoptimization of the probe concentration and labeling, and by theaveraging of the green-to-red fluorescence raweres from severalmetaphase spreads.

EXAMPLE 4

In the present study, we have used CGH to identify and map increases inDNA sequence copy number in 15 breast cancer cell lines and 33uncultured primary breast tumors.

MATERIALS AND METHODS

DNA samples. Fifteen breast cancer cell lines (3T-20, BT-474, BT-483,MCF7, MDA-157, MDA-175, MDA-231, MDA-330, MDA-361, MDA435, MDA436,MDA453, SK-BR-3, ZR-75-1, ZR-75-30) we re obtained from American TypeCulture Collection (Rockville, Md.). The cells were grown in therecommended culture conditions in 75 cm² flasks until confluent. Thetyypsinized cells were suspended in a digestion buffer (0.1 mg/mlproteinases K, 100 mM NaC., 10 mM Tris-Ci pH 8, 25 mM EDTA pH 8, 0.5%sodium dodecyl sulfate) and were incubated with shaping at 50° C.overnight. High-molecular weight DNA was extracted usingPhenolCmoroform-isoamyl alcohol and precipitated with 7.5 M ammoniurnacetate and 100% ethanol. DNA also isolated from thirty-three primarybreast carcinomas obtained prospectively at surgery. Thirty of thecarcinomas were ductal invasive, 1 was intraductal and 2 lobular. Thepost-operative TNM-stage distribution was stage I (6 cases), stage IIa(13 cases), stage IIb (8 cases), stage III (3 cases), stage IV (1 case)and unknown (1 case). DNA was also isolated from the peripheral blood of7 normal healthy individuals. One of these was used as the normalreference DNA in all CGH hybridizations.

Comparative Genomic Hybridizatin. The target metaphase slides wereprepared from PHA-stimulated peripheral blood lymphocytes from a normalmale. To assess the hybridization characteristics, each batch of slideswas extensively tested with labeled normal genomic DNA and withwhole-chromosome painting probes. If evidence of dim or nonuniformhybridization was detected, the entire batch of slides was abandoned anda new batch was prepared.

CGH was performed essentially as described above. Briefly, DNA sampleswere labeled either with biotin-14-dATP (test samples) ordigoxigenin-11-dUTP (normal reference DNA) using the Bionick LabelingSystems (BRL, Gaithersburgh Md.). The amount of DNAse and DNA polymeraseI was adjusted so that the probe fragment size distribution afterlabeling was 600-2000 bps (a smear in a non-denaturing agarose gel).Probe fragments of this size were necessary to obtain uniform, intensehybridization. Sixty to 100 ng of each of the labeled probes and 5 μg ofunlabeled Cot-1 DNA were precipitated with ethanol. The DNAs weredissolved in 10 μl of hybridization buffer (50% formamide, 10% dextransulfate, 2×SSC, pH 7). Metaphase slidles were denatured in 70%formanide, 2×SSC (pH 7) at 70° C. for 3 minutes, dehydrated in 70%, 85%and 100% ethanol, treated with proteinase K (0.1 μg/ml in 20 mM Tris, 2mM CaCl, pH 7.5) at 37° C. for 7.5 min and dehydrated again. Thehybridization mixture was applied on slides and hybridized for 2-3 daysat 37° C. in a moist chamber.

After hybridization, the slides were washed three times in wash buffer(50% formamide, 2×SSC, pH 7), twice in 2×SSC and once in 0.1×SSC at 45°C. for 10 min each. Biotinylated DNA was detected with 5μg/mlAvidin-FITC (green fluorescence) and digoxigenin-labeled DNA with 1μg/ml anti-digoxigenin Rhodamine (red fluorescence). Only one layer ofimmunoreagents was used as this was found to reduce noise and provide amore uniform fluorescence signal. After staining, the slides were washedin 4×SSC, 4×SSC/0.1% Triton-X, and 4×SSC for 10 min each. Samples werecounterstained with 4,6-diamidino-2-phenylindole (DAPI) in a antifadesolution.

Digital imnaee analysis. The hybridizations were analyzed using adigital image analys is system that was based on either a Nikon SA orZeiss Axioplan microscope equipped with a cooled CCD camera PhotometricsInc. Tucson, Ariz.), and a filter system consisting of a tripleband passbeam splitter and emission fiters. Excitation of e ach fluorochrome wasaccomplished using single band pass excitation filters in a computercontrolled filter wheel. This made it possible to collect sequential,properly registered imag es of the mthre fluorochromes o API, FITC, andRhodamine). The three-color images were processed with a Sun IPXworkstation using Scil-Image software (TNO, Delft, Netherlands) forpseudo-color display. Contrmst-stretched three-color images were used tovisually inspect the color change along the metaphase chromosomes. Acontrast-stretched single color image of the DAPI counterstain was usedto identify chromosomes and assign copy number changes to individualchromosomal bands.

In a ddition to visual analysis of the digital images, a quantitativeanalysis of green and red fluorescence intensities was performed withXwooiz software as described in Kalioniemi, A., Kafiooniemi, O. -P.,Sudar, D., Rutovitz, D., Gray, J. W., Waldman, F & Pinkel, D, (1992)Science 258, 818-821. Kallioniemi, O. -P., Kalrioniemi, A., Sudar, D.,Rutovitz, D., Gray, J. W., Waldman, F. & Pinkel, D. (1993) Semin. CancerBiol. 4, 41-46. The contour and medial axis of chromosomes were definedbased on the DAPI counterstain. Local background fluorescence wasdetermined for each chromosome and subtracted from the green and redimages before analysis. Green and red fluorescence intensities were thendetermined along the chromosome from p-telomere to q-telomere byintegrating fluorescence across the width of the chromosomeperpendicular to the medial axis. Green and red fluorescence intensityratio profiles were then calculated for each chromosome. All profileswere normalized so that the overall green to red ratio for the entiremetaphase (within the segmented image of DAPI and red fluorescence) wasset at 1.0.

Interpretation of CGH images. Five metaphases from each hybridizationwere analyzed for the chromosomal locations of DNA sequence increases.These regions were determined using green to red fluorescence intensityratio profiles and information gained during visual inspection of thedigital images. Criteria used to define the increased DNA sequence copynumber in tumors were based on comparisons of normal DNAs labeled andstained with two different colors. These included: green to red ratiosthat exceeded 1.25 or small paired spots of green fluorescence clearlyabove the background level found in the normal vs. normal DNAcomparisons (see below). High-level increases were defined as thosechromosomal subregions where the green to red ratio exceeded 1.75.Increases that were not systematically present in all metaphases or thatwere seen only in one chromatid or in one of the two chromosomehomologues were considered non-specific and were excluded from analysis.

RESULTS

Interpretation of CGH data was guided by control experiments.Comparisons among seven normal DNA specimens were used to establishnormal levels of green to red fluorescence intensity ratio variationalong the length of all human chromosomes while cell lines with knownamplifications were used to assess sensitivity. FIour of 6 breast cancercell lines with known ERBB2 amplification and 3 of 5 with known BCL1amplification showed evidence of increased copy number by CGH at 17q12and 1 1q13, as expected. All high level amplifications (5-15×) weredetected by CGH, while those of a lower level (2-5×) were missed. Nofalse positive ERBB2 or BCL1 amplifications were seen.

Twenty-eight (85%) of the 33 primary breast carcinomas showed evidenceof increased DNA sequence copy number involving one or more regions ofthe genome (FIGS. 14-16). Twenty-one cases (64%) showed gains of wholechromosomes or chromosome arms and 20 (61%) showed increases in copynumber involving only a region of an arm. All 15 breast cancer celllines showed copy number increases, 14 of them (93%) whole chromosome orchromosome arm gains and 14 (93%) region copy number increases (FIG. 16,Table 4). The average number of changes per specimen was much higher incell lines (7.7±3.3) than in primary tumors (3.3±2.6).

The most common copy number changes were remarkably similar in bothprimary tumors and cell lines. Gains of whole chromosome arms were mostoften found at 1q (36% of primary tumors/40% of cell lines) and at 8q(27%/40%), while regional copy number increases were seen repeatedly at17q22-24 (18%/67%) and at 20q13 (18%/40%). The region on chromosome 17was distinctly distal to the ERBB2 gene locus and was amplifiedindependently of it. Increases in copy number at 17q22-q24 and 20q13 inBTA474, MCF7, and MDA-361 cell lines were validated using fluorescencein situ hybridization with specific probes to these loci.

DNA sequence copy number increases were also found in a large number ofother chromosomal regions, but at a lower frequency (Table 5). Overall,a total of 26 loci (15 in primary tumors and 19 in cell lines) appearedto be significant as they were involved in either 1) high-level copynumber increase of a small chromosomal segment, or 2) low-level regionalincreases that occurred in at least three primary tumors (>9% of cases)or two (<13%) cell lines. Usually, several regions were simultaneouslyamplified in a single specimen.

The size distribution of the region involved in DNA sequence copy numberincreases was continuous, ranging from very small regions to whole armor whole chromosome gains.

DISCUSSION

The present results on breast cancer show how CGH provides newinformation on 1) the overall frequency of DNA gains and amplifications,2) the clustering of these changes to particular chromosomal subregions,and 3) the size and number of regions affected in individual tumors.This information also illustrates the advantages of CGH as compared toconventional molecular genetic methods that are usually restricted tothe analysis of only one locus at a time. While cytogenetic analysisdoes provide a similar overview, it is limited by technical problems inpreparing metaphase chromosomes from solid tumors, inability todetermine the genomic origin of the amplified sequences (e.g., HSRs,DMs), as well as difficulties in the unambiguous identification of allchanges in highly aberrant genomes.

Our results substantially extend the curre nt knowledge of the frequencyof DNA gains and the chromosomal regions involved in breast cancer. Thefrequently occurring whole arm gains of 1q and 8q have been previouslyreported in primaoy tumors by cytogenetic techniques, but most of theregional copy number changes, e.g. at 17q22-q24 and 20q13, have not.Loci previously known to be amplified in breast cancer (8p12, 8q24,11q13, 15q25, and 17q12) accounted for only 22% of all amplificationevents (subregional DNA s equ ence copy number increases)in the p rimaryt umors and 18% of those in the cell lines. This illustrates the factthat studies limited to known oncogene loci underestimate the level ofgenetic instability in breast cancer and provide a restricted view ofthe regions involved.

17q22-24 and 20q13 emerged as major new regions of ampli fication inbreast cancer. It is believed that these are loci where previouslyunknown genes important in breast cancer progression can be found. Theregion on 17q is telomeric to the ERBB2 and BRCA1 genes and is amplifiedindependently of ERBB2. Based on CGH analysis, this region appearsfairly large and may span may different genes (e.g. HOX2, NGFR, WNT3,GH1, GH2, PRKCA, HLR1, NME1). The region in 20q3 also contains severalknown genes, including SRC, ADA, RPN2, GNAS1 and ZNF8. Furtlier detailedstudies on 17q and 20q are necessary to determine which, if any, ofthese genes are located in the minimal common region of amplificationand expressed in brees t cancer cells.

The a bilty of CGH to detect amplification is dependent on the level ofamplification and the size of the region affected. While we have beenable to detect more than 5-7 fold amplification of oncogenes inhomogeneous cell lines (Kallioniemi, A., Kallioniemi, O.-P., Sudar, D.,Rutovitz, D., Gray, J. W., Waldman, F & Pinkel, D, (1992) Science 258,818-821. Kallioniemi, O. -P., Kallioniemi, A., Sudar, D., Rutovitz, D.,Gray, J. W., Waldman, F. & Pinkel, D. (1993) Semin. Cancer Biol. 4,41-46), the sensitivity of CGH in primary tumors is compromised bynormal cell contamination and intratumor heterogeneity. The actualamplification frequencies, especially those involving small amplicons,may therefore be higher than reported here. Furthermore, amplificationis only one of the mechanisms by which gene expression may be elevatedin tumor cells. The same gene may be amplified in some tumors andunregulated by other means in other tumors. Thus, amplificationfrequency may not directly reflect the relative importance of the genesin the various chromosomal regions, and virtually all of the 26different loci reported here may contain genes whose elevated expressioncontributes to tumor progression.

CGH not only detects and maps gains of DNA sequences, but also providesan estimate of the size of the affected region. A new finding of thisstudy was that the size distribution of the region involved in DNA gainswas virtually continuous, starting from the high-level. amplificationsof small regions (apparently ranging from a few hundred kbs to 1-2 Mbsand involving ERBB2, BCL1, and several previously unknown regions) togains of whole chromosome arms. In addition to "classical geneamplification", often described in cell lines selected for drugresistance in vitro, where usually a single target gene is activated,primary tumors more often show copy number increases of large regions(up to tens of Mbs) that may simultaneously affect the expression ofmany different genes. For example, several different regions of 8q(8q21, 8q22-q23 and 8q24) were separately amplified in a few specimens.It is possible that the very common gain of the entire long arm ofchromosome 8 is selected for because it leads to a simultaneous increaseof copy number of all these three regions. Studies of 11q13amplification in breast cancer and 12q13q-q14 amplification in sarcomassupport the concept that a single amplicon may contain several expressedsequences.

Many breast cancer cell lines and primary tumors showed simultaneousamplification of several distinct regions of the genome suggesting thatbreast cancer cells are genetically very unstable. It appears likelythat the amplification of several different genes actually contributesto growth advantage. However, co-amplification of an important oncogenein one locus and random DNA sequences from another locus cannot beexcluded.

Chromosomal regions amplified in cell lines tended to be smaller andapparently of higher copy number than those in the primary tumors. Basedon in vitro drug resistance models, the size of the amplicon tends tobecome smaller with time. The small size of the amplified regions incell lines may thereby reflect the more advanced state of theamplification process. The more advanced genetic composition of breastcancer cell lines is also evident from the much higher average number ofDNA copy number changes per specimen as compared with primary tumors.However, the chromosomal regions involved were remarkably similar. Mostof the highly prevalent changes, such as copy number increases of 8q24,11q13, 17q22-q24 and 20q13, as well as gains of 1q, 8q and 20q werefound frequently both in primary tumors and cell lines. These findingsindicate that most of the genetic aberrations present in highly-evolvedcell lines are representative of those seen in uncultured primary tumorsand that cell lines are representative of those seen in unculturedprimary tumors and that cell lines provide a valuable resource for moredetailed mapping and isolation of genes implicated in breast cancer.

                  TABLE 4                                                         ______________________________________                                        SUMMARY OF CHROMOSOMAL REGIONS INVOLVED IN DNA                                  SEQUENCE COPY NUMBER INCREASES IN 15                                          BREAST CANCER CELL LINES                                                                 Region Copy Number                                                 Cell Line Increases Whole-arm gains                                         ______________________________________                                        BT-20    4q32-q34, 6q21-q22                                                                            5p, 7p, 10p, 16q, 18p,                                   20q                                                                         BT-474 17q12, 17q22-q24, --                                                    20q13                                                                        BT-483 12q24 1q, 8q, 19q, 20q                                                 MCF7 1cen-q32, 3p14, 8q21- 5p, 12q, 14q                                        qter, 15q21-qter, 16q23-                                                      q24, 17q22-q24, 20q13                                                        MDA-157 1q32-qter, 5q32-qter, 2p, 7q, 8q, Xp                                   13q31-qter, 14q24-qter,                                                       17q22-qter, 19q13.1,                                                          19q13.4, 20q13                                                               MDA-175 11q13 1q, 8q, 20q                                                     MDA-231 -- 4p, 6p, 11q, 19q                                                   MDA-330 3p26-qter, 5q31-qter, 5p, 10p, 14q                                     7q22-q32, 8q21-q23,                                                           11p15, 11q13, 17q12,                                                          17q21-qter, 20q13.2-qter                                                     MDA-361 cen-q21, 12q21.3-q23, 5p, 8q, 12p, 16                                  12q24, 17q22-qter,                                                            19q13.3-q13.4, 20q13                                                         MDA-435 19q12-q13 3p14-qter, 8q, 20q                                          MDA436 3p22-pter, 5q31-qter, lq, 5p, 16q, 21, 22                               8q22-qter, 14q3 1-qter,                                                       17q22-qter, 20q13                                                            MDA453 1q31-qter, 3q26-qter, 8q, 14q, 20, 22                                   17q22-q24                                                                    SK-BR-3 3p22-pter, 8q21, 8q23- 1q, 7pter-q31, 16p, 20q                         q24.1, 10cen-q21,                                                             13q22-qter, 14q31,                                                            17q12, 17q24-qter                                                            ZR-75-1 11q13, 12q14-q15, 1q, 7p, 12p, 16p, 20q,                               17q22-qter 22                                                                ZR-75-30 8q23-qter, 17cen-q24 1q, 5p, 20p                                   ______________________________________                                         High-level, regional copy number increases (green to red ratios exceeding     1.75) are shown in bold.                                                 

                  TABLE 5                                                         ______________________________________                                        LOCI INVOLVED IN HIGH-LEVEL AMPLIFICATION OR                                    IN FREQUENCY LOW-LEVEL DNA SEQUENCE COPY                                      NUMBER INCREASES IN 33 PRIMARY BREAST TUMORS                                  AND 15 BREAST CANCER CELL LINES                                             ______________________________________                                        Primary tumors: 1q32, 6p23-pter, 6cen-p21.2, 6q12-q13, 7p21,                    7 cen-p12, 8q22-q23, 8q24, 11q13, 12q21, 15q24, 15q26, 17q22-q24,            19q13.3-qter 20q13                                                            Cell lines: 1q32, 3p22-pter, 3p14, 3q26-qter, 5q32-qter, 6q12-q13, 6q21,      8q21, 8q23-q24.1, 11q13, 12q21.3-q23, 12q24, 13q31-qter, 14q31,               17q12, 17q22-q24, 19q13.1, 19q13.3-qter, 20q13                               ______________________________________                                    

Loci involved in both primary tumors and cell lines are shown in bold.Whole chromosome or chromosome arm changes were excluded from analysis.

EXAMPLE 5

The CGH techique was carried out on a primary bladder cancer using themethodology set forth above. In particular, CGH was applied for theanalysis of DNA gains and losses in transformed uroepithelial cell linesand primary bladder carcinomas. There were employed five isogeneicSV40-transformed uroepithelial cell lines (n=7) previously analyzed byconventional cytogenetics and 28 primary bladder carcinomas of varyingstage and grade.

The results are reported below:

    ______________________________________                                        CGH RESULTS IN PRIMARY BLADDER CANCER (N = 28)                                     Gains (common region)                                                                          Deletions (common region)                               ______________________________________                                        18% +8q (q21)     36% -9q (q34)                                                 18% +13q (q31-qter) 29% -11p (p15)                                             29% -11q (q23-qter)                                                           25% -8p (p22-pter)                                                            25% -17p (p)                                                                  21% -3p (p21)                                                                 21% -10q (q26)                                                                21% -12q (q23-qter)                                                           21% -16p (p12-pter)                                                        ______________________________________                                    

    ______________________________________                                        CORRELATION BETWEEN CYTOGENETICS AND CGH                                        T1 high-grade cell line                                                                                 Cyto-                                               Cytogenetics CGH genetics CGH                                               ______________________________________                                        7p15-pter+        7p15-pter+                                                                              3p-   3p-                                           8q22-qter+  8q24-qter+ 4p16- 4p16-                                            9q+  9q13-qter+ 5p- 5p-                                                       * 11cen-p13+ 6q21-23-  6q21-q22                                               12+ *   15q11-q13-15q11-                                                      qter-                                                                            * 19p13.1-qter-                                                               y- *                                                                     ______________________________________                                         90% agreement other cell lines 91-100%                                   

ISOGENEIC SV40--TRANSFORMED UROEPITHELIAL CELL LINES

* All cell lines showed 5p-, 15q- and 9q+

* 8q23-q24+=>tumorigenicity

* 3p-=>high-grade

* 8q21-qter-=>high copy no.

FIG. 16 shows the gains and losses of DNA sequences in primary bladdercarcinomas.

EXAMPLE 6

The regions of common abnormality in ovarian cancer were mapped, therole of these abnormalities in ovarian cancer progression investigatedand the process of identifying the involved genes begun. This wasaccomplished through the combined analysis of loss of heterozygosity(LOH), comparative genomic hybridization (CGH) and fluorescence in situhybridization (FISH). Initial work validated our preliminary results andenabled analysis of the chromosomal changes leading to theabnormalities. Next, specific abnormalities were associated with grade.Early abnormalities may be diagnostically important and laterabnormalities may predict for metastatic disease and has been useful indisease management. Finally, the manner in which the abnormalitiesrevealed by these studies influences clinical and biological behaviorwas explored.

The structural and function gene dosage abnormalities in ovarian cancerthat result from events such as mitotic recombination leading to allelicloss, gene amplification, physical deletion, etc. were investigatedusing three techniques: LOH, FISH and CGH. CGH has been particularlyuseful since this technique allows genome wide mapping of regions ofaltered copy number (both increases and decreases from normal) in asingle experiment without prior knowledge of the locations of regions ofabnormality. Analysis of LOH complements CGH analysis since LOH detectsallelic loss or imbalance due to mitotic recombination and loss plusduplication that would be missed by CGH. FISH has proved to be usefulbecause it provides a direct measure of gene copy number in single cellsand it permits detailed mapping of regions involved in copy numberabnormalities. The number of physically mapped probes useful for FISH isalready high as a byproduct of the human genome project. In addition,development of probes specifically for use with FISH is a central goalof the recently established LBIUCSF Resource for Molecular Cytogenetics(Co-directed by the Principal Investigator of this application). FISHanalyses also provided validation for the CGH studies. For analysis ofregions with increased copy number, FISH provides information about themechanism of amplification that may be therapeutically useful. Forexample, it has been suggested that amplification in the form of doubleminutes can be eliminated by treatment with agents such as ahydroxyurea. In addition, information about the structure of theamplicon may facilitate positional cloning.

The CGH and LOH data for ovarian cancers are summarized by chromosomearm in FIG. 17 for 3C Grade III tumors. LOH was assessed at 86 separateloci. The number of different abnormal sites is remarkably large (almost11 different sites per tumor). Interestingly, the average number ofdifferent sites of abnormality per tumor in Grade I and Grade II wasonly about 3. These data reflect the dramatic genetic instability thatdevelops as tumors progress and reveal the number of differentabnormalities that occur. It is likely that these abnormalitiescontribute to tumor progression because of the strong associationbetween LOH and/or gene dosage decrease and tumor suppressor geneinactivation and between gene dosage increase and oncogene activation.Several regions of gene dosage abnormality occurred in regions of thegenome that are not associated with oncogenes or tumor suppressor genesnow known to be involved in ovarian cancer. Thus, these studies set thestage for identification of new genes that play a role in theprogression of ovarian cancer.

The concordance between LOH and CGH detection of loss was high in ourstudy (˜80% at all loci) indicating that physical deletion was the mostfrequency mechanism by which gene dosage abnormality occurred in ovariancancers. However, the concordance between our data and those of Sato isnot as high. This suggests a possible ethnic and/or environmentaldifference between ovarian cancers in the Japanese and Americanpopulations.

LOSS OF HETEROZYGOSITY. LOH was assessed in 50-surgically removedovarian cancers at 86 loci distributed over every chromosome arm (exceptfor the short arms of the acrocentric chromosomes. LOH was assessed bySouthern analysis of restriction fragment length polymorphisms (RFLP) orPCR analysis of simple sequence repeat (SSR) polymorphisms). Analyseswere carried out using DNA from each tumor and a correspondingperipheral blood sample. Details of the procedure and a description ofthe probes used are described in Yang-Feng T. L.; Han H.; Chen K. C.; LiS. B.; Claus E. B.; Carcangiu M. L.; Chambers S. K.; Chambers J. T.;Schwarts P. E. (1993) Allelic loss in ovarian cancer. Int. J. Cancer54:546-551. Yang-Feng et al; (65, Appendix 1).

Of the 50 tumors analyzed, 45 were of epithelial origin (30 serous, 4mucinous, 6 endometroid, 4 mixed mullerian and one undifferentiated), 4originated in the sexchord stroma and 1 originated in the germ line. The45 epithelial cases included 2 benign, 3 borderline, 4 Grade I, 5 GradeII and 31 Grade III or higher. The findings in this study are showngraphically by chromosome arm in FIG. 17. Regions showing consistent LOHincluded 13q (21/50), 17p(19/45), 17q (21/47) and Xp (18/44). Of course,not all loci were equally informative. For example, the locus with themost informative cases (i.e., cases showing two different alleles at theloci tested) is 13q while 8q and 12q were the regions with the fewestinformative cases. Thus, some sites of consistent LOH may have beenmissed.

COMPARATIVE GENOMIC HYBRIDIZATION. CGH is particularly useful foranalysis of solid tumors. In this procedure, dual color fluorescence insitu hybridization is performed to normal metaphase spreads usingdifferentially labeled DNA from the tumor and from a normal DNA sample.Unlabeled Cot-1 DNA is included in the hybridization mixture tocompetitively inhibit binding of repetitive sequences. DNA labeling,hybridization and probe detection is performed so that hybridized tumorDNA fluoresces green and hybridized normal DNA fluoresces red. Thechromosomes also arc stained with DAPI to facilitate chromosomeidentification. A digital imaging microscope and supporting softwaredeveloped in our laboratory is used to record three color images (red,green and blue) and to measure ratios of green to red fluorescence alongthe metaphase chromosomes. In practice, several different metaphasespreads are analyzed in each analysis, chromosome profiles arecalculated and combined to show the mean green:red ratio and standarddeviation therein for each chromosome. At present, a region isconsidered to be increased (decreased) in copy number relative to normalwhen the lower (upper) standard deviation is greater (less) than thenormal value. Thus, regions of physical gene dosage abnormalitythroughout the tumor genome are detected and mapped in a singleexperiment. Relative changes in copy number are converted to absolutecopy number changes by FISH with a few probes to unique sequences in thetumor. Studies of cell lines (600 MPE and COLO320) carrying knownregions of increased and decreased copy number suggest that CGH allowsdetection of 2- to 4-fold increases and decreases in copy number whenthe involved region is larger than 10-20 megabases. Detection of smallerregions of amplification has been achieved when the regions areamplified 5 to 10-fold.

CGH was performed on 38 of the 50 samples analyzed for LOH as describedabove. Samples analyzed using CGH included 2 benign, 3 borderline, 1Grade I, 2 Grade II and 30 Grade III. CGH analyses of 30 Grade IIItumors are summarized in FIG. 18. This figure illustrates the power ofCGH for comprehensive analysis of gene dosage abnormality. Regions ofincreased copy number and decreased copy number are detected and theextent of the region of gene dosage abnormality is mapped. The number ofchanges per tumor was 11. Abnormalities occurring in more than 30% ofthe tumors analyzed are summarized in Table 6. An immediate finding isthat tumors with amplification at 3q26 show 18 abnormalities per tumorwhile tumors that are not amplified at 3q26 show ˜6 abnormalities pertumor. Thus, amplification at 3q26 seems to be associated with geneticinstability. The data for the lower grade tumors are insufficient toallow discrimination between specific early and late lesions. However,they show only ˜3 aberrations per tumor. Patients with these tumors havea good prognosis. Thus, it will be interesting to learn whether Grade IItumors without 3q26 amplification behave clinically more like the lowergrade tumors.

                  TABLE 6                                                         ______________________________________                                        REGIONS OF FREQUENCY AMPLIFCATION AND DELETIONS                                 IN OVARIAN CANCERS DETECTED BY CGH OR LOH                                     CHROMOSOME REGION                                                                             NUMBER                                                        FEQUENCY OF CASES CGH FREQUENCY LOH                                         ______________________________________                                        INCREASED COPY No.                                                              3q26 13 42%                                                                   8q24 11 35%                                                                   DECREASED COPY No.                                                            17q11.2-q21 18 58% 45%                                                        (21/47)                                                                       17p12-p13 17 55% 42%                                                          (19/45)                                                                       1p35-1p36.1 14 45% 11%                                                        (3/27)*                                                                       Xp21 13 42% 41%                                                               (18/44)                                                                       13q14-q21.1 13 42% 42%                                                        (21/50)                                                                       8p21-p22 10 32% 26%                                                           (5/19)                                                                        16q13-q23 10 32% 23%                                                          (5/22)                                                                      ______________________________________                                         The probe tested was at 1p32.                                            

Table 6 also compares regions of genetic abnormality detection by CGHwith those detected by analysis of LOH. In general, regions found to beconsistently reduced in relative copy number also showed LOH. In fact,the concordance between LOH and reduced copy number using CGH isapproximately 80% at most loci. Chromosome 19 is an exception. Wecurrently believe that this low concordance is an artifact of CGH thatseems to be specific to chromosome 19. Future studies with FISH willaddress this issue. The high concordance overall is significant sincethe LOH and CGH studies were conducted in a blind fashion in differentlaboratories to minimize the possibility that interpretation of the CGHkaryotypes was influenced by the LOH result. This concordance suggeststhat the LOH in ovarian cancer is, for the most part, due to physicalloss of one allele. This is important since it would mean that theregions of reduced copy number detected and defined by CGH can be usedin the same way as information about LOH. That is, to indicate thelocations of inactive tumor suppressor genes. This interpretation issupported by analyses of deletions of 17p and 16q in human breast cancerusing CGH and FISH (see below) that guided efforts to positionally clonegenes involved in tumor progression.

Two other aspects of CGH and LOH data on gene dosage abnormality arealso noteworthy: 1) No single site of gene dosage abnormality occurs inall tumors. Table 6, for example, shows that the most frequent eventsoccur only in about 60% of all Grade III, serous tumors. Thus,clinically homogenous tumors are genetically heterogeneous. 2) Many ofthe imbalances are correlated within individual tumors. Strongcorrelations (p<0.1) have been observed for the aberration pairs:Xp-;3q+;13q-;8q+;8p-;17p-;17q-;8q+-;13q-;17p-;Xp- and 13q-;8p-. Theseobservations suggest a model for genetic progression like thatillustrated in FIG. 19 where the cancers progress along parallelpathways through the more-or-less serial accumulation of geneticaberrations, many of which confer the same phenotype.

Corollaries of this model are that "parallel" genetic events that conferthe same phenotype are likely to be uncorrelated while "serial" geneticevents are likely to be correlated (e.g., aberrations 1, 4 and 9 arelikely to be correlated while aberrations 1, 2 and 3 are not).

FLUORESCENCE IN SITU HYBRIDIZATION. Characterization of gene dosageabnormality using CGH or by analysis of LOH provide information aboutevents that occur in most or all cells in the tumor population. Inaddition, both are limited in resolution to ˜10 Mb by the availabilityof polymorphic, mapped probes (for LOH) or by the organization ofmetaphase chromosomes (CGH). FISH with well mapped probes complementsLOH and CGH by providing higher resolution mapping, information aboutheterogeneity, level of amplification information about mechanisms ofamplification. We have performed several studies illustrating the use ofFISH as an adjunct to CGH and LOH.

Amplification: CGH studies suggest frequent amplification of sequencesat 3q26 and 8q24 and deletions of 3p. We applied dual color FISH withprobes to these regions to confirm these findings and to provide moredetailed information about the level of amplification. This experimentconfirms the deletion of 3p and amplification at 3q. In addition, itextends the information available from CGH since it gives a moreaccurate estimate of the level of amplification. We also carried outhybridization with CMYC to a tumor detected as amplified at 8q24 usingCGH. The results confirm that CMYC is amplified in these tumors. We haveextended these studies by applying FISH with CMYC to other ovariantumors showing no amplification levels of CMYC amplification. Thesestudies show substantial heterogeneity in the level of amplificationamong cells within each tumor as well as amplification in low frequencysubpopulations in tumors that do not appear amplified by othertechniques. A similar result was obtained during analysis of Her-2/neuamplification in breast cancers. Information about low frequency,genetically aberrant subpopulations may be useful in defining thebiological importance of gene dosage abnormality since thesesubpopulations may confer advantage to the subpopulation that may withtime, affect the biological and/or clinical behavior of the tumor. Wealso applied FISH with probes to CMYC to short term cultures of highgrade ovarian tumors to determine whether amplification wasintrachromosomal or extrachromosomal. So far, most tumors showamplification as inter chromosomal suggesting that efforts to purgeamplified sequences chemotherapeutically will be unsuccessful. However,additional work will be required to determine the chromosomalorganization of other amplified sequences and to determine whetherchromosome integration occurs late in tumor development.

Positional cloning: The utility of FISH as an adjunct to CGH andanalysis of LOH for definition of regions of gene dosage abnormality isillustrated by our work defining regions of reduced and increased copynumber in breast cancer. These regions of gene dosage abnormality wereinitially recognized in CGH studies. Our approach has been to definethese regions by application of FISH with probes distributed at fewmegabase intervals over the regions of interest. Probes for thesestudies were acquired as anonymous cosmid clones from the NationalLaboratory Gene Library Project and mapped with 2-3 Mb precision usingour custom built semi-automatic digital imaging microscope. Generationof high density probes for mapping studies is straightforward with thissystem since probes can be mapped at the rate of a few per day.

The region 20q13-20qter appeared to be consistently increased in copynumber in breast cancer cell lines and in primary breast cancers. Wehave applied FISH with mapped probes to define the region and nature ofamplification more precisely. Amplification was defined simply bycounting the number of hybridization signals in interphase nuclei ateach locus for which a mapped probe existed. Previous studies ofHer-2/neu amplification indicated that this gives an accurate estimateof the level of amplification. FIG. 20a shows the collection of probesmapped and used for this purpose. FIG. 20b shows the level ofamplification along chromosome 20 in the breast cancer cell line BT474.The level of amplification varied significantly as a function ofposition along the chromosome suggesting a very complex amplificationprocess with the maximum (>40fold amplification) occurring in a 2 Mbregion at 20q13.2. This localized the amplified region substantially andsuggested a study in metaphase to investigate the mechanism by which theamplification occurred. The results of metaphase analysis show at leasttwo different amplicon structures. Information about amplicon structureis important since it may identify simple amplicons that will facilitatepositional cloning efforts (e.g., microdissection and cloning ofsequences from one insertion site may define a particularly simpleamplicon).

FISH with mapped probes also has proved useful in localizing theinactive genes that may have tumor suppressor function. Chromosome 16qappears to be consistently present in reduced copy number by CGH andshowing LOH in cancers of the breast, prostate, bladder and ovary. Wehave applied FISH to characterize 1(q in breast cancer using physicallymapped cosmid probes. In these studies, dual color FISH was performedusing a probe to the test locus and to the centromere of chromosome 16.Two patterns of hybridization were observed, one where the ratio of thenumber of signals from the test probe was about the same as the numberof signals from the centromere probes and one where the test signal tocentromere signal ratio was less than one. Interestingly, the latterpattern correlates strongly with LOH while the former signal does not.This is also true for chromosome 17 near the p53 locus. Thus, at theseloci, LOH appears to be caused by physical deletion of one alleleindicating the location of an inactive tumor suppressor gene. Thesestudies suggested 16q22.1 as the location of that gene. The celladhesion molecule E-cadherin maps to that region. Thus, we selected aprobe to E-cadherin and used this to explore E-cadherin deletions in 11breast cancer cell lines. A high correlation was observed betweendeletion of one E-adherin allele and loss of E-cadherin expression.However, the FISH studies indicate that one allele of E-adherin remainedin all lines. This suggested that the remaining allele of E-cadherinmust be mutated. We tested this by analyzing the 16 exons of E-cadherinat the DNA sequence level using nonisotopic SSCP analysis and by DNAsequencing. All cell lines showing loss of E-cadherin expression showedinactivating mutations. The cell lines expressing E-cadherin appearednormal at the DNA sequence level. Thus, loss of cell adhesion leading toincreased local invasion and/or metastasis is suggested as an importantevent involved in the progression of breast cancer. FISH was useful inlocalizing this inactivated gene since inactivation occurred by physicalloss of one allele. Since ovarian cancers also show reduced copy numberat 16q, it is reasonable to determine whether E-cadherin inactivationplays a role in the progression of this cancer as well.

The descriptions of the foregoing embodiments of the invention have beenpresented for purposes of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formdisclosed, and obviously many modifications and variations are possiblein light of the above teachings. The embodiments were chosen anddescribed in order to best explain the principles of the invention andits practical application to enable thereby others skilled in the art tobest utilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated. It isintended that the scope of the invention be defined by the claimsappended hereto. All references cited herein are incorporated byreference.

We claim:
 1. A method of detecting a chromosomal abnormality in asuspected bladder cancer sample by detecting an amplification of uniquesequences of at least one position selected from the group consisting ofq21 on human chromosome 8, q31-qter on human chromosome 13, p15-pter onhuman chromosome 7, q24-qter on human chromosome 8, cen-p13 on humanchromosome 11 and q13-qter on human chromosome 9, in a genome beingtested, said method comprising the steps of:(a) differently labellingDNA sequences from the test genome and a normnal human genome; (b)hybridizing said labelled DNA sequences from each of said genomes to areference genome under the following conditions:(i) either the labelledDNA sequences or the reference genome, or both, have their repetitivesequences blocked and/or removed; and (ii) unique DNA sequences in thereference genome are retained; and (c) comparing the intensities of thesignals from the labelled DNA sequences as a function of position on thereference genome, thereby allowing detection of the presence or absenceof the amplification in the test genome.
 2. The method of claim 1,wherein the step of comparing the intensities of the signals from thelabelled DNA sequences comprises determining the ratio of theintensities of the signals as a function of position in the referencegenome.
 3. The method of claim 1, wherein the amplification is detectedat position q21 of human chromosome 8 in the test genome.
 4. The methodof claim 1, wherein the amplification is detected at position q31-qterof human chromosome 13 in the test genome.
 5. The method of claim 1,wherein the amplification is detected at position p15-pter on humanchromosome 7 in the test genome.
 6. The method of claim 1, wherein theamplification is detected at position q24-qter on human chromosome 8 inthe test genome.
 7. The method of claim 1, wherein the amplification isdetected at position q13-qter on human chromosome 9 in the test genome.8. The method of claim 1, wherein the reference genome comprises atleast one metaphase chromosome.
 9. A method of detecting a chromosomalabnormality in a suspected bladder cancer sample by detecting a deletionof a unique sequence of at least one position selected from the groupconsisting of q34 on human chromosome 9, p22-pter on human chromosome 8,the p arm of human chromosome 17, p21 on human chromosome 3, the p armof human chromosome 9, q26 on human chromosome 10, q23-qter on humanchromosome 12, p12-pter on human chromosome 16, the p arm of humanchromosome 3, p16 on human chromosome 4, the p arm of human chromosome5, q21-q22 on human chromosome 6, q11-q13 on human chromosome 15,q21-q23 on human chromosome 6, p15 on human chromosome 11, q23-qter onhuman chromosome 11, q11-qter on human chromosome 15 and p13.1-pter onhuman chromosome 19, in a genome being tested, said method comprisingthe steps of:(a) differently labelling DNA sequences from the test,genome and a normal human genome; (b) hybridizing said labelled DNAsequences from each of said genomes to a reference genome under thefollowing conditions:(i) either the labelled DNA sequences or thereference genome, or both, have their repetitive sequences blockedand/or removed; and (ii) unique DNA sequences in the reference genomeare retained; and (c) comparing the intensities of the signals from thelabelled DNA sequences as a function of position on the referencegenome, thereby allowing detection of the presence or absence of thedeletion in the test genome.
 10. The method of claim 9, wherein the stepof comparing the intensities of the signals from the labelled DNAsequences comprises determining the ratio of the intensities of thesignals as a function of position in the reference genome.
 11. Themethod of claim 9, wherein the deletion is detected at position q34 ofhuman chromosome
 9. 12. The method of claim 9, wherein the deletion isdetected at position p22-pter of human chromosome
 8. 13. The method ofclaim 9, wherein the deletion is detected on the p arm of humanchromosome
 17. 14. The method of claim 9, wherein the deletion isdetected at position p21 on human chromosome
 3. 15. The method of claim9, wherein the deletion is detected on the p arm of human chromosome 9.16. The method of claim 9, wherein the deletion is detected at positionq26 on human chromosome
 10. 17. The method of claim 9, wherein thedeletion is detected at position q23-qter of human chromosome
 12. 18.The method of claim 9, wherein the deletion is detected at positionp12-pter of human chromosome
 16. 19. The method of claim 9, wherein thedeletion is detected on the p arm of human chromosome
 3. 20. The methodof claim 9, wherein the deletion is detected at position p16 on humanchromosome
 4. 21. The method of claim 9, wherein the deletion isdetected on the p arm of human chromosome
 5. 22. The method of claim 9,wherein the deletion is detected at position q21-q22 on human chromosome6.
 23. The method of claim 9, wherein the deletion is detected atposition q11-q13 on human chromosome
 15. 24. The method of claim 9,wherein the deletion is detected at position p13.1-qter on humanchromosome
 19. 25. The method of claim 9, wherein the reference genomecomprises at least one metaphase chromosome.
 26. A method for detectinga chromosomal abnormality in a suspected bladder cancer sample bydetecting an amplification of unique sequences of at least one positionselected from the group consisting of q21 on human chromosome 8, andq31-qter on human chromosome 13, said method comprising the stepsof:contacting a chromosome sample from a patient with a compositionconsisting essentially of one or more labeled nucleic acid probes eachof which binds selectively to a target polynucleotide sequence of atleast one position selected from the group consisting of q21 on humanchromosome 8, and q31-qter on human chromosome 13, under conditions inwhich the probe forms a stable hybridization complex with the targetsequence; anddetecting the hybridization complex, thereby detecting thepresence or absence of the amplification.
 27. The method of claim 26,wherein the amplification is detected at position q21 of humanchromosome
 8. 28. The method of claim 26, wherein the amplification isdetected at position q31-qter of human chromosome
 13. 29. A method ofdetecting a chromosomal abnormality in a suspected bladder cancer sampleby detecting a deletion of a unique sequence of at least one positionselected from the group consisting of q23-qter on human chromosome 12,p12-pter on human chromosome 16, p16 on human chromosome 4, q11-q13 onhuman chromosome 15, q23-qter on human chromosome 11, q11-qter on humanchromosome 15 and p13.1-pter on human chromosome 19, said methodcomprising the steps of:contacting a chromosome sample from a patientwith a composition consisting essentially of one or more labeled nucleicacid probes each of which binds selectively to a target polynucleotidesequence of at least one position selected from the group consisting ofq23-qter on human chromosome 12, p12-pter on human chromosome 16, p16 onhuman chromosome 4, q11-q13 on human chromosome 15, q23-qter on humanchromosome 11, q11-qter on human chromosome 15 and p13.1-pter on humanchromosome 19, under conditions in which the probe forms a stablehybridization complex with the target sequence; and detecting thehybridization complex, thereby detecting the presence or absence of thedeletion.
 30. The method of claim 29, wherein the deletion is detectedat position q23-qter of human chromosome
 12. 31. The method of claim 29,wherein the deletion is detected at position p12-pter of humanchromosome
 16. 32. The method of claim 29, wherein the deletion isdetected at position p16 on human chromosome
 4. 33. The method of claim29, wherein the deletion is detected at position q11-q13 on humanchromosome
 15. 34. The method of claim 29, wherein the deletion isdetected at position p13.1-pter on human chromosome
 19. 35. A method fordetecting bladder cancer in a patient comprising detecting anamplification of unique sequences of at least one position selected fromthe group consisting of q21 on human chromosome 8, and q31-qter on humanchromosome 13, or a deletion of a unique sequence of at least oneposition selected from the group consisting of q23-qter on humanchromosome 12, p12-pter on human chromosome 16, p16 on human chromosome4, q11-q13 on human chromosome 15, q23-qter on human chromosome 11,q11-ter on human chromosome 15 and p13.1-pter on human chromosome 19,said method comprising the steps of:contacting a chromosome sample froma patient with a composition consisting essentially of one or morelabeled nucleic acid probes each of which binds selectively to a targetpolynucleotide sequence of at least one position selected from the groupconsisting of q21 on human chromosome 8, and q31-qter on humanchromosome 13, or a deletion of a unique sequence of at least oneposition selected from the group consisting of q23-qter on humanchromosome 12, p12-pter on human chromosome 16, p16 on human chromosome4, q11-q13 on human chromosome 15, q23-qter on human chromosome 11,q11-qter on human chromosome 15 and p13.1-pter on human chromosome 19,under conditions in which the probe forms a stable hybridization complexwith the target sequence; and detecting the hybridization complex,thereby detecting the presence or absence of the amplification ordeletion.
 36. The method of claim 1, wherein the amplification isdetected at position cen-p13 on human chromosome
 11. 37. The method ofclaim 9, wherein the deletion is detected at position q21-q23 on humanchromosome
 6. 38. The method of claim 9 wherein the deletion is detectedat position p15 on human chromosome
 11. 39. The method of claim 9wherein the deletion is detected at position q23-qter on humanchromosome
 11. 40. The method of claim 9 wherein the deletion isdetected at position q11-qter on human chromosome
 15. 41. The method ofclaim 29 wherein the deletion is detected at position q23-qter on humanchromosome
 11. 42. The method of claim 28 wherein the deletion isdetected at position q11-qter on human chromosome 15.